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1.
Exp Eye Res ; 217: 108977, 2022 04.
Artigo em Inglês | MEDLINE | ID: mdl-35139333

RESUMO

Loss-of-function mutations in the Wnt co-receptor, low-density lipoprotein receptor-related protein 5 (LRP5), result in familial exudative vitreoretinopathy (FEVR), osteoporosis-pseudoglioma syndrome (OPPG), and Norrie disease. CRISPR/Cas9 gene editing was used to produce rat strains deficient in Lrp5. The purpose of this study was to validate this rat model for studies of hypovascular, exudative retinopathies. The retinal vasculature of wildtype and Lrp5 knockout rats was stained with Giffonia simplifolia isolectin B4 and imaged by fluorescence microscopy. Effects on retinal structure were investigated by histology. The integrity of the blood-retina barrier was analyzed by measurement of permeability to Evans blue dye and staining for claudin-5. Retinas were imaged by fundus photography and SD-OCT, and electroretinograms were recorded. Lrp5 gene deletion led to sparse superficial retinal capillaries and loss of the deep and intermediate plexuses. Autofluorescent exudates were observed and are correlated with increased Evans blue permeability and absence of claudin-5 expression in superficial vessels. OCT images show pathology similar to OCT of humans with FEVR, and retinal thickness is reduced by 50% compared to wild-type rats. Histology and OCT reveal that photoreceptor and outer plexiform layers are absent. The retina failed to demonstrate an ERG response. CRISPR/Cas9 gene-editing produced a predictable rat Lrp5 knockout model with extensive defects in the retinal vascular and neural structure and function. This rat model should be useful for studies of exudative retinal vascular diseases involving the Wnt and norrin pathways.


Assuntos
Proteína-5 Relacionada a Receptor de Lipoproteína de Baixa Densidade , Retina , Animais , Claudina-5/biossíntese , Claudina-5/genética , Azul Evans/farmacologia , Vitreorretinopatias Exsudativas Familiares/genética , Vitreorretinopatias Exsudativas Familiares/metabolismo , Proteína-5 Relacionada a Receptor de Lipoproteína de Baixa Densidade/genética , Proteína-5 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Mutação , Ratos , Retina/metabolismo , Relação Estrutura-Atividade
2.
PLoS Genet ; 15(5): e1008130, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-31048900

RESUMO

Nanophthalmos is a rare, potentially devastating eye condition characterized by small eyes with relatively normal anatomy, a high hyperopic refractive error, and frequent association with angle closure glaucoma and vision loss. The condition constitutes the extreme of hyperopia or farsightedness, a common refractive error that is associated with strabismus and amblyopia in children. NNO1 was the first mapped nanophthalmos locus. We used combined pooled exome sequencing and strong linkage data in the large family used to map this locus to identify a canonical splice site alteration upstream of the last exon of the gene encoding myelin regulatory factor (MYRF c.3376-1G>A), a membrane bound transcription factor that undergoes autoproteolytic cleavage for nuclear localization. This variant produced a stable RNA transcript, leading to a frameshift mutation p.Gly1126Valfs*31 in the C-terminus of the protein. In addition, we identified an early truncating MYRF frameshift mutation, c.769dupC (p.S264QfsX74), in a patient with extreme axial hyperopia and syndromic features. Myrf conditional knockout mice (CKO) developed depigmentation of the retinal pigment epithelium (RPE) and retinal degeneration supporting a role of this gene in retinal and RPE development. Furthermore, we demonstrated the reduced expression of Tmem98, another known nanophthalmos gene, in Myrf CKO mice, and the physical interaction of MYRF with TMEM98. Our study establishes MYRF as a nanophthalmos gene and uncovers a new pathway for eye growth and development.


Assuntos
Glaucoma de Ângulo Fechado/genética , Hiperopia/genética , Proteínas de Membrana/genética , Microftalmia/genética , Degeneração Retiniana/genética , Fatores de Transcrição/genética , Adulto , Animais , Criança , Pré-Escolar , Éxons , Família , Feminino , Mutação da Fase de Leitura/genética , Variação Genética/genética , Glaucoma de Ângulo Fechado/metabolismo , Humanos , Hiperopia/metabolismo , Masculino , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microftalmia/metabolismo , Pessoa de Meia-Idade , Linhagem , Sítios de Splice de RNA/genética , Erros de Refração/genética , Fatores de Transcrição/metabolismo
3.
J Biol Chem ; 295(14): 4647-4660, 2020 04 03.
Artigo em Inglês | MEDLINE | ID: mdl-32086377

RESUMO

Vascular endothelial growth factor (VEGF) contributes to blood-retinal barrier (BRB) dysfunction in several blinding eye diseases, including diabetic retinopathy. Signaling via the secreted protein norrin through the frizzled class receptor 4 (FZD4)/LDL receptor-related protein 5-6 (LRP5-6)/tetraspanin 12 (TSPAN12) receptor complex is required for developmental vascularization and BRB formation. Here, we tested the hypothesis that norrin restores BRB properties after VEGF-induced vascular permeability in diabetic rats or in animals intravitreally injected with cytokines. Intravitreal co-injection of norrin with VEGF completely ablated VEGF-induced BRB permeability to Evans Blue-albumin. Likewise, 5-month diabetic rats exhibited increased permeability of FITC-albumin, and a single norrin injection restored BRB properties. These results were corroborated in vitro, where co-stimulation of norrin with VEGF or stimulation of norrin after VEGF exposure restored barrier properties, indicated by electrical resistance or 70-kDa RITC-dextran permeability in primary endothelial cell culture. Interestingly, VEGF promoted norrin signaling by increasing the FZD4 co-receptor TSPAN12 at cell membranes in an MAPK/ERK kinase (MEK)/ERK-dependent manner. Norrin signaling through ß-catenin was required for BRB restoration, but glycogen synthase kinase 3 α/ß (GSK-3α/ß) inhibition did not restore BRB properties. Moreover, levels of the tight junction protein claudin-5 were increased with norrin and VEGF or with VEGF alone, but both norrin and VEGF were required for enriched claudin-5 localization at the tight junction. These results suggest that VEGF simultaneously induces vascular permeability and promotes responsiveness to norrin. Norrin, in turn, restores tight junction complex organization and BRB properties in a ß-catenin-dependent manner.


Assuntos
Barreira Hematorretiniana/metabolismo , Permeabilidade Capilar/efeitos dos fármacos , Proteínas do Olho/farmacologia , Fator A de Crescimento do Endotélio Vascular/farmacologia , Animais , Barreira Hematorretiniana/efeitos dos fármacos , Bovinos , Claudina-5/metabolismo , Diabetes Mellitus Experimental/induzido quimicamente , Diabetes Mellitus Experimental/patologia , Masculino , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Ratos , Ratos Long-Evans , Retina/metabolismo , Vasos Retinianos/citologia , Vasos Retinianos/metabolismo , Transdução de Sinais/efeitos dos fármacos , Tetraspaninas/genética , Tetraspaninas/metabolismo , Regulação para Cima/efeitos dos fármacos , beta Catenina/antagonistas & inibidores , beta Catenina/metabolismo
4.
J Neuroinflammation ; 18(1): 186, 2021 Aug 26.
Artigo em Inglês | MEDLINE | ID: mdl-34446062

RESUMO

BACKGROUND: Several retinal pathologies exhibit both inflammation and breakdown of the inner blood-retinal barrier (iBRB) resulting in vascular permeability, suggesting that treatments that trigger resolution of inflammation may also promote iBRB restoration. METHODS: Using the mouse retinal ischemia-reperfusion (IR) injury model, we followed the time course of neurodegeneration, inflammation, and iBRB disruption and repair to examine the relationship between resolution of inflammation and iBRB restoration and to determine if minocycline, a tetracycline derivative shown to reverse microglial activation, can hasten these processes. RESULTS: A 90-min ischemic insult followed by reperfusion in the retina induced cell apoptosis and inner retina thinning that progressed for approximately 2 weeks. IR increased vascular permeability within hours, which resolved between 3 and 4 weeks after injury. Increased vascular permeability coincided with alteration and loss of endothelial cell tight junction (TJ) protein content and disorganization of TJ protein complexes. Shunting of blood flow away from leaky vessels and dropout of leaky capillaries were eliminated as possible mechanisms for restoring the iBRB. Repletion of TJ protein contents occurred within 2 days after injury, long before restoration of the iBRB. In contrast, the eventual re-organization of TJ complexes at the cell border coincided with restoration of the barrier. A robust inflammatory response was evident a 1 day after IR and progressed to resolution over the 4-week time course. The inflammatory response included a rapid and transient infiltration of granulocytes and Ly6C+ classical inflammatory monocytes, a slow accumulation of Ly6Cneg monocyte/macrophages, and activation, proliferation, and mobilization of resident microglia. Extravasation of the majority of CD45+ leukocytes occurred from the superficial plexus. The presence of monocyte/macrophages and increased numbers of microglia were sustained until the iBRB was eventually restored. Intervention with minocycline to reverse microglial activation at 1 week after injury promoted early restoration of the iBRB coinciding with decreased expression of mRNAs for the microglial M1 markers TNF-α, IL-1ß, and Ptgs2 (Cox-2) and increased expression of secreted serine protease inhibitor Serpina3n mRNA. CONCLUSIONS: These results suggest that iBRB restoration occurs as TJ complexes are reorganized and that resolution of inflammation and restoration of the iBRB following retinal IR injury are functionally linked.


Assuntos
Barreira Hematorretiniana/patologia , Inflamação/patologia , Traumatismo por Reperfusão/patologia , Retina/patologia , Vasos Retinianos/patologia , Animais , Apoptose/fisiologia , Permeabilidade Capilar/fisiologia , Fragmentação do DNA , Modelos Animais de Doenças , Camundongos , Microglia/metabolismo , Recuperação de Função Fisiológica/fisiologia
5.
J Immunol ; 202(4): 1057-1068, 2019 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-30635390

RESUMO

Autoimmune retinopathy (AIR) is a treatable condition that manifests in acute and progressive vision loss in patients. It has recently been determined that AIR is associated with an imbalance of TH1 versus regulatory T cell immunity toward the retinal protein, recoverin. This study describes a new murine model to understand the immunopathology of AIR and its association with T cell responses toward recoverin. Immunization of C57BL/6 mice with recoverin resulted in ocular inflammation including infiltration of CD4+ and CD8+ T lymphocytes, B cells, and CD11b+Ly6C+ inflammatory monocytes in the eyes. Production of IFN-γ and IL-17 from T cells was exacerbated in IL-10 knockout (KO) mice and kinetics of disease development was accelerated. Infiltration of T cells and inflammatory monocytes into the eyes dramatically increased in recoverin-immunized IL-10 KO mice. An immunodominant peptide of recoverin, AG-16, was capable of inducing disease in IL-10 KO mice and resulted in expansion of AG-16 tetramer-specific CD4+ T cells in lymphoid organs and eyes. Adoptive transfer of recoverin-stimulated cells into naive mice was sufficient to induce AIR, and immunization of B cell-deficient mice led to a milder form of the disease. This model supports the hypothesis that recoverin-specific T cell responses are major drivers of AIR pathogenesis and that IL-10 is an important factor in protection.


Assuntos
Doenças Autoimunes/imunologia , Olho/imunologia , Interleucina-10/imunologia , Recoverina/imunologia , Doenças Retinianas/imunologia , Animais , Olho/patologia , Inflamação/imunologia , Interleucina-10/deficiência , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Monócitos/imunologia , Células Th1/imunologia , Células Th17/imunologia
6.
Bioorg Med Chem ; 28(11): 115480, 2020 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-32327351

RESUMO

Studies demonstrate that small molecule targeting of atypical protein kinase C (aPKC) may provide an effective means to control vascular permeability, prevent edema, and reduce inflammation providing novel and important alternatives to anti-VEGF therapies for certain blinding eye diseases. Based on a literature tricyclic thieno[2,3-d]pyrimidine lead (1), an ATP-competitive inhibitor of the aPKC iota (ι) and aPKC zeta (ζ) isoforms, we have synthesized a small series of compounds in 1-2 steps from a readily available chloro intermediate. A single pyridine congener was also made using 2D NMR to assign regiochemistry. Within the parent pyrimidine series, a range of potencies was observed against aPKCζ whereas the pyridine congener was inactive. Selected compounds were also tested for their effect toward VEGF-induced permeability in BREC cells. The most potent of these (7l) was further assayed against the aPKCι isoform and showed a favorable selectivity profile against a panel of 31 kinases, including kinases from the AGC superfamily, with a focus on PKC isoforms and kinases previously shown to affect permeability. Further testing of 7l in a luciferase assay in HEK293 cells showed an ability to prevent TNF-α induced NFκB activation while not having any effect on cell survival. Intravitreal administration of 7l to the eye yielded a complete reduction in permeability in a test to determine whether the compound could block VEGF- and TNFα-induced permeability across the retinal vasculature in a rat model. The compound in mice displayed good microsomal stability and in plasma moderate exposure (AUC and Cmax), low clearance, a long half-life and high oral bioavailability. With IV dosing, higher levels were observed in the brain and eye relative to plasma, with highest levels in the eye by either IV or PO dosing. With a slow oral absorption profile, 7l accumulates in the eye to maintain a high concentration after dosing with higher levels than in plasma. Compound 7l may represent a class of aPKC inhibitors for further investigation.


Assuntos
Citocinas/antagonistas & inibidores , Edema/tratamento farmacológico , Proteína Quinase C/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Pirimidinas/farmacologia , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Animais , Citocinas/metabolismo , Relação Dose-Resposta a Droga , Edema/induzido quimicamente , Edema/metabolismo , Feminino , Células HEK293 , Humanos , Camundongos , Estrutura Molecular , Proteína Quinase C/metabolismo , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/química , Pirimidinas/síntese química , Pirimidinas/química , Ratos , Ratos Long-Evans , Relação Estrutura-Atividade , Fator A de Crescimento do Endotélio Vascular/metabolismo
7.
Am J Pathol ; 188(10): 2392-2405, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30220554

RESUMO

Changes in permeability of retinal blood vessels contribute to macular edema and the pathophysiology of numerous ocular diseases, including diabetic retinopathy, retinal vein occlusions, and macular degeneration. Vascular endothelial growth factor (VEGF) induces retinal permeability and macular thickening in these diseases. However, inflammatory agents, such as tumor necrosis factor-α (TNF-α), also may drive vascular permeability, specifically in patients unresponsive to anti-VEGF therapy. Recent evidence suggests VEGF and TNF-α induce permeability through distinct mechanisms; however, both require the activation of atypical protein kinase C (aPKC). We provide evidence, using genetic mouse models and therapeutic intervention with small molecules, that inhibition of aPKC prevented or reduced vascular permeability in animal models of retinal inflammation. Expression of a kinase-dead aPKC transgene, driven by a vascular and hematopoietic restricted promoter, reduced retinal vascular permeability in an ischemia-reperfusion model of retinal injury. This effect was recapitulated with a small-molecule inhibitor of aPKC. Expression of the kinase-dead aPKC transgene dramatically reduced the expression of inflammatory factors and blocked the attraction of inflammatory monocytes and granulocytes after ischemic injury. Coinjection of VEGF with TNF-α was sufficient to induce permeability, edema, and retinal inflammation, and treatment with an aPKC inhibitor prevented VEGF/TNF-α-induced permeability. These data suggest that aPKC contributes to inflammation-driven retinal vascular pathology and may be an attractive target for therapeutic intervention.


Assuntos
Permeabilidade Capilar/fisiologia , Proteína Quinase C/antagonistas & inibidores , Vasos Retinianos/fisiologia , Animais , Permeabilidade Capilar/efeitos dos fármacos , Masculino , Camundongos Endogâmicos C57BL , Papiledema/induzido quimicamente , Papiledema/fisiopatologia , Ratos Long-Evans , Proteínas Recombinantes , Traumatismo por Reperfusão/fisiopatologia , Retinite/induzido quimicamente , Retinite/fisiopatologia , Junções Íntimas/química , Junções Íntimas/fisiologia , Fator de Necrose Tumoral alfa/farmacologia , Fator A de Crescimento do Endotélio Vascular/farmacologia
8.
Sensors (Basel) ; 16(3): 269, 2016 Feb 23.
Artigo em Inglês | MEDLINE | ID: mdl-26907297

RESUMO

An attachable electromagnetic-energy-harvester driven wireless vibration-sensing system for monitoring milling-processes and cutter-wear/breakage-conditions is demonstrated. The system includes an electromagnetic energy harvester, three single-axis Micro Electro-Mechanical Systems (MEMS) accelerometers, a wireless chip module, and corresponding circuits. The harvester consisting of magnets with a coil uses electromagnetic induction to harness mechanical energy produced by the rotating spindle in milling processes and consequently convert the harnessed energy to electrical output. The electrical output is rectified by the rectification circuit to power the accelerometers and wireless chip module. The harvester, circuits, accelerometer, and wireless chip are integrated as an energy-harvester driven wireless vibration-sensing system. Therefore, this completes a self-powered wireless vibration sensing system. For system testing, a numerical-controlled machining tool with various milling processes is used. According to the test results, the system is fully self-powered and able to successfully sense vibration in the milling processes. Furthermore, by analyzing the vibration signals (i.e., through analyzing the electrical outputs of the accelerometers), criteria are successfully established for the system for real-time accurate simulations of the milling-processes and cutter-conditions (such as cutter-wear conditions and cutter-breaking occurrence). Due to these results, our approach can be applied to most milling and other machining machines in factories to realize more smart machining technologies.

9.
J Neuroinflammation ; 10: 149, 2013 Dec 10.
Artigo em Inglês | MEDLINE | ID: mdl-24325836

RESUMO

BACKGROUND: Many retinal diseases are associated with vascular dysfunction accompanied by neuroinflammation. We examined the ability of minocycline (Mino), a tetracycline derivative with anti-inflammatory and neuroprotective properties, to prevent vascular permeability and inflammation following retinal ischemia-reperfusion (IR) injury, a model of retinal neurodegeneration with breakdown of the blood-retinal barrier (BRB). METHODS: Male Sprague-Dawley rats were subjected to 45 min of pressure-induced retinal ischemia, with the contralateral eye serving as control. Rats were treated with Mino prior to and following IR. At 48 h after reperfusion, retinal gene expression, cellular inflammation, Evan's blue dye leakage, tight junction protein organization, caspase-3 activation, and DNA fragmentation were measured. Cellular inflammation was quantified by flow-cytometric evaluation of retinal tissue using the myeloid marker CD11b and leukocyte common antigen CD45 to differentiate and quantify CD11b+/CD45low microglia, CD11b+/CD45hi myeloid leukocytes and CD11bneg/CD45hi lymphocytes. Major histocompatibility complex class II (MHCII) immunoreactivity was used to determine the inflammatory state of these cells. RESULTS: Mino treatment significantly inhibited IR-induced retinal vascular permeability and disruption of tight junction organization. Retinal IR injury significantly altered mRNA expression for 21 of 25 inflammation- and gliosis-related genes examined. Of these, Mino treatment effectively attenuated IR-induced expression of lipocalin 2 (LCN2), serpin peptidase inhibitor clade A member 3 N (SERPINA3N), TNF receptor superfamily member 12A (TNFRSF12A), monocyte chemoattractant-1 (MCP-1, CCL2) and intercellular adhesion molecule-1 (ICAM-1). A marked increase in leukostasis of both myeloid leukocytes and lymphocytes was observed following IR. Mino treatment significantly reduced retinal leukocyte numbers following IR and was particularly effective in decreasing the appearance of MHCII+ inflammatory leukocytes. Surprisingly, Mino did not significantly inhibit retinal cell death in this model. CONCLUSIONS: IR induces a retinal neuroinflammation within hours of reperfusion characterized by inflammatory gene expression, leukocyte adhesion and invasion, and vascular permeability. Despite Mino significantly inhibiting these responses, it failed to block neurodegeneration.


Assuntos
Barreira Hematorretiniana/efeitos dos fármacos , Inflamação/patologia , Minociclina/farmacologia , Fármacos Neuroprotetores/farmacologia , Traumatismo por Reperfusão/patologia , Retina/patologia , Animais , Barreira Hematorretiniana/patologia , Permeabilidade Capilar/efeitos dos fármacos , Imunofluorescência , Expressão Gênica/efeitos dos fármacos , Masculino , Degeneração Neural/patologia , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase em Tempo Real , Retina/efeitos dos fármacos
10.
Biochem J ; 446(3): 455-67, 2012 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-22721706

RESUMO

Pro-inflammatory cytokines and growth factors such as VEGF (vascular endothelial growth factor) contribute to the loss of the BRB (blood-retinal barrier) and subsequent macular oedema in various retinal pathologies. VEGF signalling requires PKCß [conventional PKC (protein kinase C)] activity; however, PKCß inhibition only partially prevents VEGF-induced endothelial permeability and does not affect pro-inflammatory cytokine-induced permeability, suggesting the involvement of alternative signalling pathways. In the present study, we provide evidence for the involvement of aPKC (atypical PKC) signalling in VEGF-induced endothelial permeability and identify a novel class of inhibitors of aPKC that prevent BRB breakdown in vivo. Genetic and pharmacological manipulations of aPKC isoforms were used to assess their contribution to endothelial permeability in culture. A chemical library was screened using an in vitro kinase assay to identify novel small-molecule inhibitors, and further medicinal chemistry was performed to delineate a novel pharmacophore. We demonstrate that aPKC isoforms are both sufficient and required for VEGF-induced endothelial permeability. Furthermore, these specific, potent, non-competitive, small-molecule inhibitors prevented VEGF-induced tight junction internalization and retinal endothelial permeability in response to VEGF in both primary culture and in rodent retina. The results of the present study suggest that aPKC inhibition with 2-amino-4-phenyl-thiophene derivatives may be developed to preserve the BRB in retinal diseases such as diabetic retinopathy or uveitis, and the BBB (blood-brain barrier) in the presence of brain tumours.


Assuntos
Barreira Hematorretiniana/metabolismo , Proteína Quinase C/antagonistas & inibidores , Inibidores de Proteínas Quinases/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Animais , Bovinos , Retinopatia Diabética/metabolismo , Humanos , Masculino , Isoformas de Proteínas/antagonistas & inibidores , Isoformas de Proteínas/metabolismo , Proteína Quinase C/metabolismo , Ratos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Retina/citologia , Retina/metabolismo , Vasos Retinianos/citologia , Vasos Retinianos/metabolismo , Transdução de Sinais , Fator A de Crescimento do Endotélio Vascular/genética
11.
Front Cell Neurosci ; 17: 1192464, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37377777

RESUMO

Introduction: Clinical trials demonstrated that co-targeting angiopoietin-2 (Ang-2) and vascular endothelial growth factor (VEGF-A) with faricimab controls anatomic outcomes and maintains vision improvements, with strong durability, through 2 years in patients with neovascular age-related macular degeneration and diabetic macular edema. The mechanism(s) underlying these findings is incompletely understood and the specific role that Ang-2 inhibition plays requires further investigation. Methods: We examined the effects of single and dual Ang-2/VEGF-A inhibition in diseased vasculatures of JR5558 mice with spontaneous choroidal neovascularization (CNV) and in mice with retinal ischemia/reperfusion (I/R) injuries. Results: In JR5558 mice, Ang-2, VEGF-A, and dual Ang-2/VEGF-A inhibition reduced CNV area after 1 week; only dual Ang-2/VEGF-A inhibition decreased neovascular leakage. Only Ang-2 and dual Ang-2/VEGF-A inhibition maintained reductions after 5 weeks. Dual Ang-2/VEGF-A inhibition reduced macrophage/microglia accumulation around lesions after 1 week. Both Ang-2 and dual Ang-2/VEGF-A inhibition reduced macrophage/microglia accumulation around lesions after 5 weeks. In the retinal I/R injury model, dual Ang-2/VEGF-A inhibition was statistically significantly more effective than Ang-2 or VEGF-A inhibition alone in preventing retinal vascular leakage and neurodegeneration. Discussion: These data highlight the role of Ang-2 in dual Ang-2/VEGF-A inhibition and indicate that dual inhibition has complementary anti-inflammatory and neuroprotective effects, suggesting a mechanism for the durability and efficacy of faricimab in clinical trials.

12.
Commun Biol ; 6(1): 408, 2023 04 13.
Artigo em Inglês | MEDLINE | ID: mdl-37055505

RESUMO

Studies of primary visual cortex have furthered our understanding of amblyopia, long-lasting visual impairment caused by imbalanced input from the two eyes during childhood, which is commonly treated by patching the dominant eye. However, the relative impacts of monocular vs. binocular visual experiences on recovery from amblyopia are unclear. Moreover, while sleep promotes visual cortex plasticity following loss of input from one eye, its role in recovering binocular visual function is unknown. Using monocular deprivation in juvenile male mice to model amblyopia, we compared recovery of cortical neurons' visual responses after identical-duration, identical-quality binocular or monocular visual experiences. We demonstrate that binocular experience is quantitatively superior in restoring binocular responses in visual cortex neurons. However, this recovery was seen only in freely-sleeping mice; post-experience sleep deprivation prevented functional recovery. Thus, both binocular visual experience and subsequent sleep help to optimally renormalize bV1 responses in a mouse model of amblyopia.


Assuntos
Ambliopia , Córtex Visual , Masculino , Animais , Camundongos , Ambliopia/terapia , Acuidade Visual , Privação Sensorial/fisiologia , Córtex Visual/fisiologia , Modelos Animais de Doenças , Sono
13.
Invest Ophthalmol Vis Sci ; 62(10): 20, 2021 08 02.
Artigo em Inglês | MEDLINE | ID: mdl-34410299

RESUMO

Purpose: Diabetic retinopathy results in vision loss with changes to both retinal blood vessels and neural retina. Recent studies have revealed that animal models of diabetes demonstrate early loss of visual function. We explored the time course of retinal change in three different mouse models of diabetes in a longitudinal study using in vivo measures of retinal structure (optical coherence tomography [OCT]) and visual function (optomotor and pupillary responses). Methods: OCT analysis of retinal microstructure, optokinetic response as a measure of visual acuity, and pupillary response to light stimulation were compared among the db/db, Ins2Akita, and streptozotocin (STZ)-induced mouse models of diabetes at 1.5, 3, 6, and 9 months of diabetes. Results: The db/db, Ins2Akita, and STZ-induced models of diabetes all exhibited vision loss and retinal thinning as disease progressed. Both structural changes and functional measures were significantly correlated with the blood glucose levels. Despite this, vision loss and retinal thinning were not consistently correlated, except for the inner retinal layer thickness at 6 months of diabetes. Conclusions: This longitudinal study compiled structural measures and functional outcome data for type 1 and 2 diabetes mouse models commonly used for diabetes studies and demonstrated an overall decline in retinal-related health in conjunction with weight change and blood glucose alterations. The relationship between the structural change and functional outcome could be correlative but is not necessarily causative, as retinal thinning was not sufficient to explain visual acuity decline.


Assuntos
Diabetes Mellitus Experimental/diagnóstico , Retinopatia Diabética/patologia , Retina/patologia , Vasos Retinianos/patologia , Tomografia de Coerência Óptica/métodos , Acuidade Visual/fisiologia , Animais , Diabetes Mellitus Experimental/fisiopatologia , Retinopatia Diabética/fisiopatologia , Seguimentos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Retina/fisiopatologia , Vasos Retinianos/fisiopatologia
14.
Diabetes ; 70(7): 1549-1560, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-33883214

RESUMO

Diabetic retinopathy is one of the leading causes of vision loss and blindness. Extensive preclinical and clinical evidence exists for both vascular and neuronal pathology. However, the relationship of these changes in the neurovascular unit and impact on vision remains to be determined. Here, we investigate the role of tight junction protein occludin phosphorylation at S490 in modulating barrier properties and its impact on visual function. Conditional vascular expression of the phosphorylation-resistant Ser490 to Ala (S490A) form of occludin preserved tight junction organization and reduced vascular endothelial growth factor (VEGF)-induced permeability and edema formation after intraocular injection. In the retinas of streptozotocin-induced diabetic mice, endothelial-specific expression of the S490A form of occludin completely prevented diabetes-induced permeability to labeled dextran and inhibited leukostasis. Importantly, vascular-specific expression of the occludin mutant completely blocked the diabetes-induced decrease in visual acuity and contrast sensitivity. Together, these results reveal that occludin acts to regulate barrier properties downstream of VEGF in a phosphorylation-dependent manner and that loss of inner blood-retinal barrier integrity induced by diabetes contributes to vision loss.


Assuntos
Barreira Hematorretiniana/fisiologia , Diabetes Mellitus Experimental/fisiopatologia , Retinopatia Diabética/fisiopatologia , Ocludina/fisiologia , Acuidade Visual , Animais , Leucostasia/prevenção & controle , Camundongos , Camundongos Endogâmicos C57BL , Permeabilidade , Fosforilação , Estreptozocina , Fator A de Crescimento do Endotélio Vascular/fisiologia
15.
J Ophthalmic Vis Res ; 15(4): 446-452, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33133434

RESUMO

PURPOSE: To evaluate the penetration of carbon nanotubes (CNTs) throughout retinoblastoma in a transgenic mice model. METHODS: CNTs functionalized with fluorescein isothiocyanate and targeting ligands biotin (CTN-FITC-Bio, 0.5mg/ml), or folic acid (CNT-FITC-FA, 0.5mg/ml) were injected into the vitreous of one eye of LH BETA T AG transgenic mice. Other eye did not receive any injection and was used as control. Three mice were sacrificed at days 1, 2, and 3. Eyes were enucleated and stained with 4,6-diamidino-2-phenylindole. The sections were imaged by fluorescent microscope. The images were transformed into grey-scale in MATLAB for intensity analysis. Background intensity was normalized by marking squares outside the eyeball and using the mean intensity of these squares. Fluorescent intensity (FI) for each image was measured by calculating the intensity of a same-sized square within retinoblastoma. RESULTS: Nine eyes of nine mice were included in each CNT-FITC-Bio and CNT-FITC-FA groups. The mean FI in CNT-FITC-Bio was 52.08 ± 6.33, 53.62 ± 9.00, and 65.54 ± 5.14 in days 1, 2, and 3, respectively. The mean FI in CNT-FITC-FA was 50.28 ± 7.37, 59.21 ± 6.43, and 58.38 ± 2.32 on days 1, 2, and 3, respectively. FI was significantly higher in eyes injected with CNT-FITC-Bio and CNT-FITC-FA compared to the control eyes (P = 0.02). There was no difference in FI between eyes with CNT-FITC-Bio and CNT-FITC-FA, and FI remained stable on days 1-3 in CNT-FITC-Bio, CNT-FITC-FA, and control eyes (P > 0.05). CONCLUSION: We observed higher FI in eyes with CNT-FITC-Bio and CNT-FITC-FA compared to control eyes, showing penetration of CNTs throughout retinoblastoma. CNTs can be a carrier candidate for imaging or therapeutic purposes in retinoblastoma.

16.
Invest Ophthalmol Vis Sci ; 61(6): 8, 2020 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-32492112

RESUMO

Purpose: Extracellular accumulation of all-trans-retinaldehyde (atRAL), a highly reactive visual cycle intermediate, is toxic to cells of the outer retina and contributes to retinal and macular degenerations. However, the contribution of atRAL to retinal capillary function has not been studied. We hypothesized that atRAL released from the outer retina can contribute to retinal vascular permeability. We, therefore, tested the contribution of atRAL to retinal ischemia-reperfusion (IR)-induced vascular permeability. Methods: IR was induced in mice by transient increase in intraocular pressure followed by natural reperfusion. The visual cycle was ablated in the Lrat-/- mice, reduced by dark adaptation or the use of the RPE65 inhibitor and atRAL scavenger emixustat. Accumulation of FITC-BSA was used to assess vascular permeability and DNA fragmentation quantified cell death after IR. Primary bovine retinal endothelial cell (BREC) culture was used to measure the direct effects of atRAL on endothelial permeability and cell death. Results: Inhibition of the visual cycle by Lrat-/-, dark adaptation, or with emixustat, all reduced approximately half of IR induced vascular permeability at 48 hours. An increase in BREC permeability with atRAL coincided with lactate dehydrogenase (LDH) release, a measure of cell death. Both permeability and toxicity were blocked by emixustat. Conclusions: Outer retinal pathology may contribute to vascular permeability by release of atRAL, which can act directly on vascular endothelial cells to alter barrier properties and induce cell death. These studies may have implications for a variety of blinding eye diseases that include outer retinal damage and retinal vascular permeability.


Assuntos
Permeabilidade Capilar/fisiologia , Traumatismo por Reperfusão/metabolismo , Vasos Retinianos/metabolismo , Retinaldeído/fisiologia , Animais , Bovinos , Morte Celular , Fragmentação do DNA , Adaptação à Escuridão , Impedância Elétrica , Células Endoteliais/efeitos dos fármacos , Ensaio de Imunoadsorção Enzimática , Pressão Intraocular/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Estresse Oxidativo/efeitos dos fármacos , Éteres Fenílicos/farmacologia , Propanolaminas/farmacologia , cis-trans-Isomerases/antagonistas & inibidores
17.
Sci Rep ; 9(1): 881, 2019 01 29.
Artigo em Inglês | MEDLINE | ID: mdl-30696927

RESUMO

Diabetic peripheral neuropathy (DPN), diabetic kidney disease (DKD), and diabetic retinopathy (DR) contribute to significant morbidity and mortality in diabetes patients. The incidence of these complications is increasing with the diabetes epidemic, and current therapies minimally impact their pathogenesis in type 2 diabetes (T2D). Improved mechanistic understanding of each of the diabetic complications is needed in order to develop disease-modifying treatments for patients. We recently identified fundamental differences in mitochondrial responses of peripheral nerve, kidney, and retinal tissues to T2D in BKS-db/db mice. However, whether these mitochondrial adaptations are the cause or consequence of tissue dysfunction remains unclear. In the current study BKS-db/db mice were treated with the mitochondrial uncoupler, niclosamide ethanolamine (NEN), to determine the effects of mitochondrial uncoupling therapy on T2D, and the pathogenesis of DPN, DKD and DR. Here we report that NEN treatment from 6-24 wk of age had little effect on the development of T2D and diabetic complications. Our data suggest that globally targeting mitochondria with an uncoupling agent is unlikely to provide therapeutic benefit for DPN, DKD, or DR in T2D. These data also highlight the need for further insights into the role of tissue-specific metabolic reprogramming in the pathogenesis of diabetic complications.


Assuntos
Diabetes Mellitus Tipo 2/metabolismo , Proteínas de Desacoplamento Mitocondrial/metabolismo , Animais , Nefropatias Diabéticas/metabolismo , Neuropatias Diabéticas/metabolismo , Retinopatia Diabética/metabolismo , Modelos Animais de Doenças , Etanolamina/farmacologia , Rim/metabolismo , Masculino , Camundongos , Mitocôndrias/metabolismo , Proteínas de Desacoplamento Mitocondrial/fisiologia , Niclosamida/farmacologia , Desacopladores/farmacologia
18.
Invest Ophthalmol Vis Sci ; 57(6): 2584-92, 2016 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-27163772

RESUMO

PURPOSE: Inflammation associated with blood-retinal barrier (BRB) breakdown is a common feature of several retinal diseases. Therefore, the development of novel nonsteroidal anti-inflammatory approaches may provide important therapeutic options. Previous studies demonstrated that inhibition of dipeptidyl peptidase-IV, the enzyme responsible for the degradation of glucagon-like peptide-1 (GLP-1), led to insulin-independent prevention of diabetes-induced increases in BRB permeability, suggesting that incretin-based drugs may have beneficial pleiotropic effects in the retina. In the current study, the barrier protective and anti-inflammatory properties of exendin-4 (Ex-4), an analog of GLP-1, after ischemia-reperfusion (IR) injury were examined. METHODS: Ischemia-reperfusion injury was induced in rat retinas by increasing the intraocular pressure for 45 minutes followed by 48 hours of reperfusion. Rats were treated with Ex-4 prior to and following IR. Blood-retinal barrier permeability was assessed by Evans blue dye leakage. Retinal inflammatory gene expression and leukocytic infiltration were measured by qRT-PCR and immunofluorescence, respectively. A microglial cell line was used to determine the effects of Ex-4 on lipopolysaccharide (LPS)-induced inflammatory response. RESULTS: Exendin-4 dramatically reduced the BRB permeability induced by IR injury, which was associated with suppression of inflammatory gene expression. Moreover, in vitro studies showed that Ex-4 also reduced the inflammatory response to LPS and inhibited NF-κB activation. CONCLUSIONS: The present work suggests that Ex-4 can prevent IR injury-induced BRB breakdown and inflammation through inhibition of inflammatory cytokine production by activated microglia and may provide a novel option for therapeutic intervention in diseases involving retinal inflammation.


Assuntos
Barreira Hematorretiniana/efeitos dos fármacos , Peptídeo 1 Semelhante ao Glucagon/análogos & derivados , Inflamação/prevenção & controle , Isquemia/prevenção & controle , Peptídeos/farmacologia , Traumatismo por Reperfusão/complicações , Doenças Retinianas/prevenção & controle , Peçonhas/farmacologia , Animais , Bovinos , Células Cultivadas , Modelos Animais de Doenças , Exenatida , Immunoblotting , Imuno-Histoquímica , Incretinas/farmacologia , Inflamação/metabolismo , Isquemia/etiologia , Isquemia/metabolismo , Masculino , Ratos , Ratos Long-Evans , Traumatismo por Reperfusão/metabolismo , Doenças Retinianas/etiologia , Doenças Retinianas/metabolismo
19.
Int J Pharm ; 480(1-2): 15-26, 2015 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-25575473

RESUMO

In this study, a multiparticulate pulsatile drug delivery system activated by a rupturable controlled-release membrane (Eudragit(®) RS) via osmotic pressure (with NaCl as the osmogent) was developed and characterized for omeprazole, omeprazole sodium, and propranolol HCl which have different water solubilities. Multiparticulates in pellet form for incorporation with or without the osmogent were manufactured by three methods and then used to coat a polymeric membrane. Results demonstrated that drug/osmogent-containing pellets manufactured by the extrusion/spheronization method with incorporation of the osmogent were optimal. The lag time (tL) to initiate pulsatile release is regulated by tL=l(2)/(6×D), which is dependent on the coating levels (l(2)) and plasticizer content (D). The pulsatile release pattern was found to be dependent on the osmotic pressure (osmogent), drug solubility, and mechanical properties of the polymeric membrane (elasticity and toughness). Omeprazole with lower water solubility could not generate sufficient osmotic pressure to create a crack in the membrane to activate pulsatile release, whereas the two other model drugs with higher solubilities could. But adsorption of omeprazole sodium on Eudragit(®) RS via charge-charge interactions led the its incomplete release. Finally, with 4% osmogent of NaCl added, a lag time in a range from 0 to 12h proportionally regulated by varying both the membrane thickness and plasticizer level initiated the complete pulsatile release of propranolol HCl. In conclusion, a multiparticulate pulsatile drug delivery system activated by a rupturable controlled-release membrane via osmotic pressure was successfully developed, and clinical applications of chronotherapy with drugs like propranolol HCl are expected.


Assuntos
Sistemas de Liberação de Medicamentos , Omeprazol/administração & dosagem , Pressão Osmótica , Propranolol/administração & dosagem , Resinas Acrílicas/química , Química Farmacêutica/métodos , Preparações de Ação Retardada , Composição de Medicamentos/métodos , Liberação Controlada de Fármacos , Excipientes/química , Omeprazol/química , Polímeros/química , Propranolol/química , Solubilidade , Fatores de Tempo
20.
J Gastrointest Surg ; 6(3): 379-86, 2002.
Artigo em Inglês | MEDLINE | ID: mdl-12022990

RESUMO

L-Arginine, which is intimately involved in cellular immune functions and nitric oxide biology, is transported by intestinal cells largely via transport System y(+). The gut epithelium is exposed to various luminal amino acids at any given time, and therefore the purpose of this study was to study the regulation of luminal arginine transport by other amino acids. System y(+) L-arginine transport activity was measured in Caco-2 monolayers exposed to various amino acids. L-arginine and/or other System y(+) substrates specifically upregulated System y(+) transport activity twofold after 1 hour, with a response noted as early as 5 minutes. Non-System y(+) substrates did not affect L-arginine absorption. Kinetic analysis indicated that L-arginine exposure increased both System y(+) K(m) and V(max). Neither cycloheximide nor actinomycin affected this stimulation, indicating that the regulation did not involve transcription or translation. The System y(+) substrate activation effect was reversible. L-arginine transport activity returned to baseline within 3 hours when cells were reincubated in amino acid-free media. These data indicate that System y(+) arginine transport activity is rapidly and reversibly activated by System y(+) substrates via a mechanism consistent with transmembrane stimulation. These findings identify a mechanism by which luminal nutrients regulate arginine uptake by the gut.


Assuntos
Sistema y+ de Transporte de Aminoácidos/fisiologia , Arginina/metabolismo , Células CACO-2/metabolismo , Adaptação Fisiológica , Transporte Biológico , Células Cultivadas , Humanos
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