RESUMO
Although the pathogenesis of choroidal neovascularization (CNV) is largely unknown in age-related macular degeneration (AMD), inflammasomes may contribute to CNV development and progression. To understand the role NLRP3 inflammasomes in CNV, we used Ccr2RFPCx3cr1GFP dual-reporter mice and immunostaining techniques to confirm localization of NLRP3 inflammasomes in the laser-induced CNV (LCNV) lesions. Confocal microscopy was used to image and quantify LCNV volumes. MCC950 was used as NLRP3 inhibitor. ELISA and quantitative RT-PCR were used to confirm the activation of NLRP3 by monitoring the expression of IL-1ß protein and mRNA in choroidal tissues from LCNV mice. In addition, NLRP3 (-/-) LCNV mice were used to investigate whether NLRP3 inflammasomes contribute to the development of LCNV lesions. We observed that red fluorescent protein (RFP)-positive monocyte-derived macrophages and GFP-positive microglia-derived macrophages, in addition to other cell types, were localized in LCNV lesions at day 7 post-laser injury. In addition, NLRP3 inflammasomes are associated with LCNV lesions. Inhibition of NLRP3 inflammasomes, using MCC950, caused an increased Ccr2RFP-positive macrophages, Cx3cr1GFP-positive microglia, and other cells, resulting in an increase in total lesion size. NLRP3 (-/-) LCNV mice showed significantly increased lesion size compared with age-matched controls. Inhibition of NLRP3 resulted in decreased IL-1ß mRNA and protein expression in the choroidal tissues, suggesting that increased lesion size may not be directly related to IL-1ß.
Assuntos
Neovascularização de Coroide , Indenos , Inflamassomos , Interleucina-1beta , Microglia , Monócitos , Proteína 3 que Contém Domínio de Pirina da Família NLR , Animais , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Neovascularização de Coroide/metabolismo , Neovascularização de Coroide/patologia , Camundongos , Inflamassomos/metabolismo , Interleucina-1beta/metabolismo , Microglia/metabolismo , Monócitos/metabolismo , Camundongos Knockout , Sulfonas/farmacologia , Camundongos Endogâmicos C57BL , Furanos/farmacologia , Receptores CCR2/metabolismo , Receptores CCR2/genética , Macrófagos/metabolismo , Macrófagos/imunologia , Sulfonamidas/farmacologia , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Proteínas de Transporte/metabolismo , Proteínas de Transporte/genética , Corioide/metabolismo , Corioide/patologia , Modelos Animais de Doenças , Lasers/efeitos adversos , Degeneração Macular/patologia , Degeneração Macular/metabolismo , Degeneração Macular/genéticaRESUMO
Though the pathogenesis of choroidal neovascularization (CNV) is largely unknown in age-related macular degeneration (AMD), inflammasomes may contribute to CNV development and progression. To understand the role NLRP3 inflammasomes in CNV, we used Ccr2RFPCx3cr1GFP dual-reporter mice to characterize migration of Ccr2RFP positive monocytes and Cx3cr1GFP positive microglial cells into CNV lesions after laser-induced rupture of Bruch's membrane. MCC950 was used as NLRP3 inhibitor. Immunostaining was used to confirm localization of NLRP3 inflammasomes in the LCNV lesions. Confocal microscopy was used to image and quantify LCNV volumes. ELISA and qRT-PCR were used to confirm the activation of NLRP3 by monitoring the expression of IL-1ß protein and mRNA in choroidal tissues from LCNV mice. In addition, NLRP3 (-/-) LCNV mice were used to investigate whether NLRP3 inflammasomes contribute to the development of LCNV lesions. We observed that RFP positive monocyte-derived macrophages and GFP positive microglia-derived macrophages, in addition to other cell types, were localized in LCNV lesions at day 7 post-laser injury. In addition, NLRP3 inflammasomes are associated with LCNV lesions. Inhibition of NLRP3 inflammasomes, using MCC950, caused an increased Ccr2RFP positive macrophages, Cx3cr1GFP positive microglia, and other cells resulting in an increase in total lesion size. NLRP3 (-/-) LCNV mice, showed significantly increased lesion size compared to age-matched controls. Inhibition of NLRP3, resulted in decreased IL-1ß mRNA and protein expression in the choroidal tissues, suggesting that increased lesion size may not be directly related to IL-1ß.
RESUMO
Chronic low-grade retinal inflammation is an essential contributor to the pathogenesis of diabetic retinopathy (DR). It is characterized by increased retinal cell expression and secretion of a variety of inflammatory cytokines; among these, IL-1ß has the reputation of being a major driver of cytokine-induced inflammation. IL-1ß and other cytokines drive inflammatory changes that cause damage to retinal cells, leading to the hallmark vascular lesions of DR; these include increased leukocyte adherence, vascular permeability, and capillary cell death. Nuclear factor of activated T-cells (NFAT) is a transcriptional regulator of inflammatory cytokines and adhesion molecules and is expressed in retinal cells. Consequently, it may influence multiple pathogenic steps early in DR. We investigated the NFAT-dependency of IL-1ß-induced inflammation in human Müller cells (hMC) and human retinal microvascular endothelial cells (hRMEC). Our results show that an NFAT inhibitor, Inhibitor of NFAT-Calcineurin Association-6 (INCA-6), decreased IL-1ß-induced expression of IL-1ß and TNFα in hMC, while having no effect on VEGF, CCL2, or CCL5 expression. We also demonstrate that INCA-6 attenuated IL-1ß-induced increases of IL-1ß, TNFα, IL-6, CCL2, and CCL5 (inflammatory cytokines and chemokines), and ICAM-1 and E-selectin (leukocyte adhesion molecules) expression in hRMEC. INCA-6 similarly inhibited IL-1ß-induced increases in leukocyte adhesion in both hRMEC monolayers in vitro and an acute model of retinal inflammation in vivo. Finally, INCA-6 rescued IL-1ß-induced permeability in both hRMEC monolayers in vitro and an acute model of retinal inflammation in vivo. Taken together, these data demonstrate the potential of NFAT inhibition to mitigate retinal inflammation secondary to diabetes.
Assuntos
Retinopatia Diabética/tratamento farmacológico , Inflamação/tratamento farmacológico , Interleucina-1beta/genética , Fatores de Transcrição NFATC/genética , Vasculite Retiniana/tratamento farmacológico , Inibidores de Calcineurina/farmacologia , Células Cultivadas , Quimiocina CCL2/genética , Quimiocina CCL5/genética , Retinopatia Diabética/genética , Retinopatia Diabética/patologia , Selectina E/genética , Células Endoteliais/efeitos dos fármacos , Células Ependimogliais/efeitos dos fármacos , Células Ependimogliais/patologia , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Inflamação/genética , Inflamação/patologia , Molécula 1 de Adesão Intercelular/genética , Interleucina-1beta/farmacologia , Fatores de Transcrição NFATC/antagonistas & inibidores , Retina/efeitos dos fármacos , Retina/patologia , Vasculite Retiniana/genética , Vasculite Retiniana/parasitologia , Vasos Retinianos/efeitos dos fármacos , Vasos Retinianos/patologia , Fator de Necrose Tumoral alfa/genética , Fator A de Crescimento do Endotélio Vascular/genéticaRESUMO
Free fatty acid dysregulation in diabetics may elicit the release of inflammatory cytokines from Müller cells (MC), promoting the onset and progression of diabetic retinopathy (DR). Palmitic acid (PA) is elevated in the sera of diabetics and stimulates the production of the DR-relevant cytokines by MC, including IL-1ß, which induces the production of itself and other inflammatory cytokines in the retina as well. In this study we propose that experimental elevation of cytochrome P450 epoxygenase (CYP)-derived epoxygenated fatty acids, epoxyeicosatrienoic acid (EET) and epoxydocosapentaenoic acid (EDP), will reduce PA- and IL-1ß-induced MC inflammation. Broad-spectrum CYP inhibition by SKF-525a increased MC expression of inflammatory cytokines. Exogenous 11,12-EET and 19,20-EDP significantly decreased PA- and IL-1ß-induced MC expression of IL-1ß and IL-6. Both epoxygenated fatty acids significantly decreased IL-8 expression in IL-1ß-induced MC and TNFα in PA-induced MC. Interestingly, 11,12-EET and 19,20-EDP significantly increased TNFα in IL-1ß-treated MC. GSK2256294, a soluble epoxide hydrolase (sEH) inhibitor, significantly reduced PA- and IL-1ß-stimulated MC cytokine expression. 11,12-EET and 19,20-EDP were also found to decrease PA- and IL-1ß-induced NFκB-dependent transcriptional activity. These data suggest that experimental elevation of 11,12-EET and 19,20-EDP decreases MC inflammation in part by blocking NFκB-dependent transcription and may represent a viable therapeutic strategy for inhibition of early retinal inflammation in DR.
Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Células Ependimogliais/metabolismo , Epóxido Hidrolases/metabolismo , Ácidos Graxos/metabolismo , Neuroglia/patologia , Retinite/prevenção & controle , Células Cultivadas , Cicloexilaminas/farmacologia , Retinopatia Diabética/complicações , Células Ependimogliais/patologia , Epóxido Hidrolases/antagonistas & inibidores , Humanos , Mediadores da Inflamação/metabolismo , NF-kappa B/genética , Regiões Promotoras Genéticas , Retinite/complicações , Retinite/patologia , Triazinas/farmacologiaRESUMO
Diabetic retinopathy (DR) is triggered by retinal cell damage stimulated by the diabetic milieu, including increased levels of intraocular free fatty acids. Free fatty acids may serve as an initiator of inflammatory cytokine release from Müller cells, and the resulting cytokines are potent stimulators of retinal endothelial pathology, such as leukostasis, vascular permeability, and basement membrane thickening. Our previous studies have elucidated a role for peroxisome proliferator-activated receptor-ß/δ (PPARß/δ) in promoting several steps in the pathologic cascade in DR, including angiogenesis and expression of inflammatory mediators. Furthermore, PPARß/δ is a known target of lipid signaling, suggesting a potential role for this transcription factor in fatty acid-induced retinal inflammation. Therefore, we hypothesized that PPARß/δ stimulates both the induction of inflammatory mediators by Müller cells as well the paracrine induction of leukostasis in endothelial cells (EC) by Müller cell inflammatory products. To test this, we used the PPARß/δ inhibitor, GSK0660, in primary human Müller cells (HMC), human retinal microvascular endothelial cells (HRMEC) and mouse retina. We found that palmitic acid (PA) activation of PPARß/δ in HMC leads to the production of pro-angiogenic and/or inflammatory cytokines that may constitute DR-relevant upstream paracrine inflammatory signals to EC and other retinal cells. Downstream, EC transduce these signals and increase their synthesis and release of chemokines such as CCL8 and CXCL10 that regulate leukostasis and other cellular events related to vascular inflammation in DR. Our results indicate that PPARß/δ inhibition mitigates these upstream (MC) as well as downstream (EC) inflammatory signaling events elicited by metabolic stimuli and inflammatory cytokines. Therefore, our data suggest that PPARß/δ inhibition is a potential therapeutic strategy against early DR pathology.
Assuntos
Células Ependimogliais/efeitos dos fármacos , Leucostasia/prevenção & controle , PPAR delta/antagonistas & inibidores , PPAR beta/antagonistas & inibidores , Retinite/prevenção & controle , Sulfonas/farmacologia , Tiofenos/farmacologia , Adulto , Animais , Células Cultivadas , Citocinas/metabolismo , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Células Ependimogliais/metabolismo , Humanos , Inflamação , Leucostasia/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Ácidos Palmíticos/farmacologia , Reação em Cadeia da Polimerase em Tempo Real , Retina/efeitos dos fármacos , Retina/metabolismo , Retinite/metabolismoRESUMO
Exudative age-related macular degeneration (AMD), characterized by choroidal neovascularization (CNV), is the leading cause of irreversible blindness in developed countries. Anti-vascular endothelial growth factor (VEGF) drugs are the standard treatment for AMD, but they have limitations. Cell therapy is a promising approach for ocular diseases, and it is being developed in the clinic for the treatment of retinal degeneration, including AMD. We previously showed that subretinal injection of human umbilical tissue-derived cells (hUTCs) in a rodent model of retinal degeneration preserved photoreceptors and visual function through rescue of retinal pigment epithelial (RPE) cell phagocytosis. Here we investigated the effect of hUTCs on a rat model of laser-induced CNV and on a human RPE cell line, ARPE-19, for VEGF production. We demonstrate that subretinal injection of hUTCs significantly inhibited CNV and lowered choroidal VEGF in vivo. VEGF release from ARPE-19 decreased when co-cultured with hUTCs. Soluble VEGF receptor 1 (sVEGFR1) is identified as the only factor in hUTC conditioned medium (CM) that binds to VEGF. The level of exogenous recombinant VEGF in hUTC CM was dramatically reduced and could be recovered with sVEGFR1-neutralizing antibody. This suggests that hUTC inhibits angiogenesis through the secretion of sVEGFR1 and could serve as a novel treatment for angiogenic ocular diseases, including AMD.
RESUMO
Mouse laser-induced choroidal neovascularization (mouse LCNV) recapitulates the "wet" form of human age-related macular degeneration (AMD). Vascular cell adhesion molecule-1 (VCAM-1) is a known inflammatory biomarker, and it increases in the choroidal neovascular tissues characteristic of this experimental model. We have designed and constructed gold nanoparticles (AuNPs) functionalized with hairpin-DNA that incorporates an antisense sequence complementary to VCAM-1 mRNA (AS-VCAM-1 hAuNPs) and tested them as optical imaging probes. The 3' end of the hairpin is coupled to a near-infrared fluorophore that is quenched by the AuNP surface via Förster resonance energy transfer (FRET). Hybridization of the antisense sequence to VCAM-1 mRNA displaces the fluorophore away from the AuNP surface, inducing fluorescent activity. In vitro testing showed that hAuNPs hybridize to an exogenous complementary oligonucleotide within a pH range of 4.5-7.4, and that they are stable at reduced pH. LCNV mice received tail-vein injections of AS-VCAM-1 hAuNPs. Hyperspectral imaging revealed the delivery of AS-VCAM-1 hAuNPs to excised choroidal tissues. Fluorescent images of CNV lesions were obtained, presumably in response to the hybridization of AS-hAuNPs to LCNV-induced VCAM-1 mRNA. This is the first demonstration of systemic delivery of hAuNPs to ocular tissues to facilitate mRNA imaging of any target.
Assuntos
Neovascularização de Coroide/diagnóstico por imagem , Sondas Moleculares/administração & dosagem , RNA Mensageiro/metabolismo , Molécula 1 de Adesão de Célula Vascular/metabolismo , Degeneração Macular Exsudativa/diagnóstico por imagem , Animais , Biomarcadores/metabolismo , Corioide/irrigação sanguínea , Corioide/diagnóstico por imagem , Corioide/patologia , Corioide/efeitos da radiação , Neovascularização de Coroide/etiologia , Neovascularização de Coroide/patologia , Modelos Animais de Doenças , Corantes Fluorescentes/administração & dosagem , Corantes Fluorescentes/química , Ouro/administração & dosagem , Ouro/química , Humanos , Microscopia Intravital/métodos , Lasers/efeitos adversos , Masculino , Nanopartículas Metálicas/administração & dosagem , Nanopartículas Metálicas/química , Camundongos , Camundongos Endogâmicos C57BL , Imagem Molecular/métodos , Sondas Moleculares/química , Oligodesoxirribonucleotídeos Antissenso/administração & dosagem , Oligodesoxirribonucleotídeos Antissenso/química , Imagem Óptica/métodos , Molécula 1 de Adesão de Célula Vascular/genética , Degeneração Macular Exsudativa/etiologia , Degeneração Macular Exsudativa/patologiaRESUMO
Vascular cell adhesion molecule 1 (VCAM-1) is an important inflammatory biomarker correlating with retinal disease progression. Thus, detection of VCAM-1 mRNA expression levels at an early disease stage could be an important predictive biomarker to assess the risk of disease progression and monitoring treatment response. We have developed VCAM-1 targeted antisense hairpin DNA-functionalized gold nanoparticles (AS-VCAM-1 hAuNP) for the real time detection of VCAM-1 mRNA expression levels in retinal endothelial cells. The AS-VCAM-1 hAuNP fluorescence enhancement clearly visualized the TNF-α induced cellular VCAM-1 mRNA levels with high signal to noise ratios compared to normal serum treated cells. The scrambled hAuNP probes were minimally detectable under same image acquisition conditions. Intracellular hAuNPs were detected using transmission electron microscopy (TEM) analysis of the intact cells. In addition, the AS-VCAM-1 hAuNP probes exhibited no acute toxicity to the retinal microvascular endothelial cells as measured by live-dead assay.
Assuntos
Endotélio Vascular/metabolismo , Ouro/química , Nanopartículas Metálicas/química , RNA Mensageiro/análise , Vasos Retinianos/metabolismo , Molécula 1 de Adesão de Célula Vascular/metabolismo , Animais , Sobrevivência Celular , Células Cultivadas , DNA Antissenso/química , DNA Antissenso/genética , Endotélio Vascular/citologia , Endotélio Vascular/efeitos dos fármacos , Fluorescência , Nanopartículas Metálicas/administração & dosagem , Camundongos , Imagem Molecular/métodos , RNA Mensageiro/genética , Vasos Retinianos/citologia , Vasos Retinianos/efeitos dos fármacos , Fator de Necrose Tumoral alfa/farmacologia , Molécula 1 de Adesão de Célula Vascular/genéticaRESUMO
Purpose: To demonstrate the utility of a novel in vivo molecular imaging probe, HYPOX-4, to detect and image retinal hypoxia in real time, in a mouse model of retinal vein occlusion (RVO). Methods: Retinal vein occlusion was achieved in adult mice by photodynamic retinal vein thrombosis (PRVT). One or two major retinal vein(s) was/were occluded in close proximity to the optic nerve head (ONH). In vivo imaging of retinal hypoxia was performed using, HYPOX-4, an imaging probe developed by our laboratory. Pimonidazole-adduct immunostaining was performed and used as a standard ex vivo method for the detection of retinal hypoxia in this mouse RVO model. The retinal vasculature was imaged using fluorescein angiography (FA) and isolectin B4 staining. Retinal thickness was assessed by spectral-domain optical coherence tomography (SD-OCT) analysis. Results: By application of the standard ex vivo pimonidazole-adduct immunostaining technique, retinal hypoxia was observed within 2 hours post-PRVT. The observed hypoxic retinal areas depended on whether one or two retinal vein(s) was/were occluded. Similar areas of hypoxia were imaged in vivo using HYPOX-4. Using OCT, retinal edema was observed immediately post-PRVT induction, resolving 8 days later. Nominal preretinal neovascularization was observed at 10 to 14 days post-RVO. Conclusions: HYPOX-4 is an efficient probe capable of imaging retinal hypoxia in vivo, in RVO mice. Future studies will focus on its use in correlating retinal hypoxia to the onset and progression of ischemic vasculopathies.
Assuntos
Modelos Animais de Doenças , Fluoresceínas/administração & dosagem , Corantes Fluorescentes/administração & dosagem , Hipóxia/diagnóstico por imagem , Nitroimidazóis/administração & dosagem , Oclusão da Veia Retiniana/diagnóstico por imagem , Veia Retiniana/diagnóstico por imagem , Animais , Angiofluoresceinografia , Fluoresceínas/síntese química , Corantes Fluorescentes/síntese química , Processamento de Imagem Assistida por Computador , Edema Macular/diagnóstico , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Nitroimidazóis/síntese química , Radiossensibilizantes/administração & dosagem , Neovascularização Retiniana/diagnóstico , Tomografia de Coerência ÓpticaRESUMO
The objective of the present study was to assess the effect of elevating epoxygenated fatty acids on retinal vascular inflammation. To stimulate inflammation we utilized TNFα, a potent pro-inflammatory mediator that is elevated in the serum and vitreous of diabetic patients. In TNFα-stimulated primary human retinal microvascular endothelial cells, total levels of epoxyeicosatrienoic acids (EETs), but not epoxydocosapentaenoic acids (EDPs), were significantly decreased. Exogenous addition of 11,12-EET or 19,20-EDP when combined with 12-(3-adamantane-1-yl-ureido)-dodecanoic acid (AUDA), an inhibitor of epoxide hydrolysis, inhibited VCAM-1 and ICAM-1 expression and protein levels; conversely the diol product of 19,20-EDP hydrolysis, 19,20-DHDP, induced VCAM1 and ICAM1 expression. 11,12-EET and 19,20-EDP also inhibited leukocyte adherence to human retinal microvascular endothelial cell monolayers and leukostasis in an acute mouse model of retinal inflammation. Our results indicate that this inhibition may be mediated through an indirect effect on NFκB activation. This is the first study demonstrating a direct comparison of EET and EDP on vascular inflammatory endpoints, and we have confirmed a comparable efficacy from each isomer, suggesting a similar mechanism of action. Taken together, these data establish that epoxygenated fatty acid elevation will inhibit early pathology related to TNFα-induced inflammation in retinal vascular diseases.
Assuntos
Ácido 8,11,14-Eicosatrienoico/análogos & derivados , Compostos de Epóxi/administração & dosagem , Ácidos Graxos Insaturados/administração & dosagem , Vasculite Retiniana/tratamento farmacológico , Vasos Retinianos/citologia , Fator de Necrose Tumoral alfa/efeitos adversos , Ácido 8,11,14-Eicosatrienoico/administração & dosagem , Ácido 8,11,14-Eicosatrienoico/farmacologia , Adamantano/administração & dosagem , Adamantano/análogos & derivados , Adamantano/farmacologia , Animais , Células Cultivadas , Modelos Animais de Doenças , Regulação para Baixo , Células Endoteliais/citologia , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Compostos de Epóxi/farmacologia , Ácidos Graxos Insaturados/farmacologia , Humanos , Molécula 1 de Adesão Intercelular/genética , Molécula 1 de Adesão Intercelular/metabolismo , Ácidos Láuricos/administração & dosagem , Ácidos Láuricos/farmacologia , Masculino , Camundongos , Vasculite Retiniana/induzido quimicamente , Vasculite Retiniana/metabolismo , Vasos Retinianos/efeitos dos fármacos , Vasos Retinianos/metabolismo , Molécula 1 de Adesão de Célula Vascular/genética , Molécula 1 de Adesão de Célula Vascular/metabolismoRESUMO
Ischemia-induced hypoxia elicits retinal neovascularization and is a major component of several blinding retinopathies such as retinopathy of prematurity (ROP), diabetic retinopathy (DR) and retinal vein occlusion (RVO). Currently, noninvasive imaging techniques capable of detecting and monitoring retinal hypoxia in living systems do not exist. Such techniques would greatly clarify the role of hypoxia in experimental and human retinal neovascular pathogenesis. In this study, we developed and characterized HYPOX-4, a fluorescence-imaging probe capable of detecting retinal-hypoxia in living animals. HYPOX-4 dependent in vivo and ex vivo imaging of hypoxia was tested in a mouse model of oxygen-induced retinopathy (OIR). Predicted patterns of retinal hypoxia were imaged by HYPOX-4 dependent fluorescence activity in this animal model. In retinal cells and mouse retinal tissue, pimonidazole-adduct immunostaining confirmed the hypoxia selectivity of HYPOX-4. HYPOX-4 had no effect on retinal cell proliferation as indicated by BrdU assay and exhibited no acute toxicity in retinal tissue as indicated by TUNEL assay and electroretinography (ERG) analysis. Therefore, HYPOX-4 could potentially serve as the basis for in vivo fluorescence-based hypoxia-imaging techniques, providing a tool for investigators to understand the pathogenesis of ischemic retinopathies and for physicians to address unmet clinical needs.
Assuntos
Hipóxia/diagnóstico por imagem , Hipóxia/patologia , Imagem Óptica/métodos , Retina/diagnóstico por imagem , Retina/patologia , Doenças Retinianas/diagnóstico por imagem , Doenças Retinianas/patologia , Animais , Modelos Animais de Doenças , Corantes Fluorescentes/administração & dosagem , Microscopia Intravital/métodos , Camundongos , Neovascularização Patológica/diagnóstico por imagemRESUMO
OBJECTIVE: To determine the effect of oleic acid and linoleic acid on the production and secretion of specific diabetic retinopathy- (DR-) related cytokines: vascular endothelial growth factor (VEGF), interleukin-6 (IL-6), and interleukin-8 (IL-8) by human retinal glial cells, retinal endothelial cells, and retinal pigment epithelial cells. These expression profiles will be compared to those obtained by treatment of the same cell types with elevated D-glucose, a diabetes-relevant stimulus often used in retinal cell culture experiments. METHODS: Primary cultures of human retinal Müller cells, astrocytes, and microvascular endothelial cells (RMEC) and a human retinal pigment epithelial cell line (ARPE-19) were treated with oleic acid, linoleic acid, elevated D-glucose, or L-glucose as an osmotic control. VEGF, IL-6, and IL-8 concentrations in conditioned media were determined by colorimetric ELISA and normalized to total cellular protein. RESULTS: In the conditioned medium of human Müller cells, linoleic and oleic acid increased VEGF production by 6.4-fold and 9.9-fold, respectively. Linoleic acid also significantly increased IL-6 by 2.9-fold and IL-8 by 5.7-fold. L-glucose and D-glucose both increased VEGF by 3.1-fold in Müller cell conditioned medium. Linoleic acid increased IL-8 concentrations by 56% in human RMEC conditioned medium. Human retinal astrocytes and ARPE-19 were unaffected by all stimuli. CONCLUSIONS: Linoleic and oleic acid induce inflammatory mediators believed to be involved in the pathogenesis of diabetic retinopathy (DR). In culture, the free fatty acid insults, particularly linoleic acid, significantly increased cytokine production by Müller cells. In summary, these data identified Müller cells as the primary producer of these inflammatory mediators when treated with unsaturated fatty acids. This study also demonstrates that elevated glucose is an inadequate stimulus for assessing the production of inflammatory mediators. Therefore this study provides a novel in vitro model system of the dyslipidemia-induced inflammation occurring in DR.
RESUMO
Diabetic retinopathy (DR) is a leading cause of blindness worldwide, and its prevalence is growing. Current therapies for DR address only the later stages of the disease, are invasive, and have limited effectiveness. Retinal pericyte death is an early pathologic feature of DR. Although it has been observed in diabetic patients and in animal models of DR, the cause of pericyte death remains unknown. A novel pro-apoptotic pathway initiated by the interaction between glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and the E3 ubiquitin ligase, seven in absentia homolog 1 (Siah1), was recently identified in ocular tissues. In this article we examined the involvement of the GAPDH/Siah1 interaction in human retinal pericyte (hRP) apoptosis. HRP were cultured in 5 mm normal glucose, 25 mm l- or d-glucose for 48 h (osmotic control and high glucose treatments, respectively). Siah1 siRNA was used to down-regulate Siah1 expression. TAT-FLAG GAPDH and/or Siah1-directed peptides were used to block GAPDH and Siah1 interaction. Co-immunoprecipitation assays were conducted to analyze the effect of high glucose on the association of GAPDH and Siah1. Apoptosis was measured by Annexin V staining and caspase-3 enzymatic activity assay. High glucose increased Siah1 total protein levels, induced the association between GAPDH and Siah1, and led to GAPDH nuclear translocation. Our findings demonstrate that dissociation of the GAPDH/Siah1 pro-apoptotic complex can block high glucose-induced pericyte apoptosis, widely considered a hallmark feature of DR. Thus, the work presented in this article can provide a foundation to identify novel targets for early treatment of DR.
Assuntos
Apoptose/efeitos dos fármacos , Glucose/administração & dosagem , Gliceraldeído-3-Fosfato Desidrogenases/metabolismo , Proteínas Nucleares/metabolismo , Pericitos/efeitos dos fármacos , Retina/efeitos dos fármacos , Ubiquitina-Proteína Ligases/metabolismo , Núcleo Celular/enzimologia , Células Cultivadas , Técnicas de Silenciamento de Genes , Glucose/farmacologia , Humanos , Proteínas Nucleares/genética , Transporte Proteico , Retina/citologia , Ubiquitina-Proteína Ligases/genéticaRESUMO
PURPOSE: The peroxisome proliferator-activated receptor beta/delta (PPARß/δ) is a transcription factor with roles in metabolism, angiogenesis, and inflammation. It has yet undefined roles in retinal inflammation and diabetic retinopathy (DR). We used RNA-seq to better understand the role of the antagonist and inverse agonist of PPARß/δ, GSK0660, in TNFα-induced inflammation. Understanding the underlying mechanisms of vascular inflammation could lead to new treatments for DR. METHODS: RNA was isolated from human retinal microvascular endothelial cells treated with a vehicle, TNFα, or TNFα plus GSK0660. RNA-seq was performed with a 50 bp single read protocol. The differential expression was determined using edgeR and gene ontology, and a pathway analysis was performed using DAVID. RNA-seq validation was performed using qRT-PCR using the primers for ANGPTL4, CCL8, NOV, CXCL10, and PDPK1. RESULTS: TNFα differentially regulated 1,830 transcripts, many of which are involved in the cytokine-cytokine receptor interaction, chemokine signaling, and inflammatory response. Additionally, TNFα highly upregulated genes involved in leukocyte recruitment, including CCL5, CX3CL1, and CXCL10. GSK0660 differentially regulated 273 transcripts in TNFα-treated cells compared to TNFα alone. A pathway analysis revealed the enrichment of cytokine-cytokine receptor signaling. In particular, GSK0660 blocks the TNFα-induced upregulation of CCL8, a chemokine involved in leukocyte recruitment. CONCLUSIONS: TNFα regulates several genes related to retinal leukostasis in retinal endothelial cells. GSK0660 blocks the effect of TNFα on the expressions of cytokines involved in leukocyte recruitment, including CCL8, CCL17, and CXCL10 and it may therefore block TNFα-induced retinal leukostasis.
Assuntos
Células Endoteliais/metabolismo , Vasos Retinianos/metabolismo , Sulfonas/farmacologia , Tiofenos/farmacologia , Fator de Necrose Tumoral alfa/metabolismo , Células Cultivadas , Quimiocina CCL8/genética , Quimiocina CCL8/metabolismo , Citocinas/genética , Citocinas/metabolismo , Células Endoteliais/citologia , Células Endoteliais/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , PPAR delta/agonistas , PPAR delta/antagonistas & inibidores , PPAR beta/agonistas , PPAR beta/antagonistas & inibidores , Vasos Retinianos/citologia , Vasos Retinianos/efeitos dos fármacos , Análise de Sequência de RNA , Transdução de Sinais/efeitos dos fármacos , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
PURPOSE: Vascular endothelial growth factor (VEGF)-induced retinal vascular permeability contributes to diabetic macular edema (DME), a serious vision-threatening condition. Peroxisome proliferator-activated receptor ß/δ (PPARß/δ) antagonist/reverse agonist, GSK0660, inhibits VEGF-induced human retinal microvascular endothelial cell (HRMEC) proliferation, tubulogenesis, and oxygen-induced retinal vasculopathy in newborn rats. These VEGF-induced HRMEC behaviors and VEGF-induced disruption of endothelial cell junctional complexes may well share molecular signaling events. Thus, we sought to examine the role of PPARß/δ in VEGF-induced retinal hyperpermeability. METHODS: Transendothelial electrical resistance (TEER) measurements were performed on HRMEC monolayers to assess permeability. Claudin-1/Claudin-5 localization in HRMEC monolayers was determined by immunocytochemistry. Extracellular signal-regulated protein kinases 1 and 2 (Erk 1/2) phosphorylation, VEGF receptor 1 (VEGFR1) and R2 were assayed by Western blot analysis. Expression of VEGFR1 and R2 was measured by quantitative RT-PCR. Last, retinal vascular permeability was assayed in vivo by Evans blue extravasation. RESULTS: Human retinal microvascular endothelial cell monolayers treated with VEGF for 24 hours showed decreased TEER values that were completely reversed by the highest concentration of GSK0660 (10 µM) and PPARß/δ-directed siRNA (20 µM). In HRMEC treated with VEGF, GSK0660 stabilized tight-junctions as evidenced by Claudin-1 staining, reduced phosphorylation of Erk1/2, and reduced VEGFR1/2 expression. Peroxisome proliferator-activated receptor ß/δ siRNA had a similar effect on VEGFR expression and Claudin-1, supporting the specificity of GSK0660 in our experiments. Last, GSK0660 significantly inhibited VEGF-induced retinal vascular permeability and reduced retinal VEGFR1and R2 levels in C57BL/6 mice. CONCLUSIONS: These data suggest a protective effect for PPARß/δ antagonism against VEGF-induced vascular permeability, possibly through reduced VEGFR expression. Therefore, antagonism/reverse agonism of PPARß/δ siRNA may represent a novel therapeutic methodology against retinal hyperpermeability and is worthy of future investigation.
Assuntos
Permeabilidade Capilar/efeitos dos fármacos , Receptores Ativados por Proliferador de Peroxissomo/fisiologia , Vasos Retinianos/efeitos dos fármacos , Fator A de Crescimento do Endotélio Vascular/farmacologia , Análise de Variância , Animais , Membrana Celular/efeitos dos fármacos , Membrana Celular/fisiologia , Células Cultivadas , Claudina-1/análise , Claudina-5/análise , Impedância Elétrica , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Humanos , Imuno-Histoquímica , Sistema de Sinalização das MAP Quinases/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Modelos Animais , Receptores Ativados por Proliferador de Peroxissomo/antagonistas & inibidores , Receptores de Fatores de Crescimento do Endotélio Vascular/metabolismo , Retina/metabolismo , Sulfonas/farmacologia , Tiofenos/farmacologia , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
PURPOSE: We investigated endoglin expression in hypoxic microvascular endothelial cells and retinal endoglin expression in rats that develop experimental oxygen-induced retinopathy (OIR). We also tested neutralizing antibodies (Abs) against endoglin (anti-CD105 Ab) and VEGF (anti-VEGF Ab) either alone or in combination for efficacy against serum-induced retinal microvascular endothelial cell proliferation and retinal neovascularization (NV) in OIR rats. To our knowledge, this marks the first time that a biologic agent has been used to target retinal endoglin and modulate retinal neovascularization. METHODS: Induction of endoglin by hypoxia was measured by immunohistochemical analysis and ELISA. Proliferation was quantified using a colorimetric 5-bromo-2-deoxyuridine ELISA. Western blots were used to measure endoglin levels in retinas of OIR rats. Immunohistochemical staining was also preformed in OIR rats using anti-CD105 and fluorescein isothiocyanate-conjugated isolectin B4 antibodies. RESULTS: Anti-CD105 Ab and Anti-VEGF Ab, administered alone or in combination, reduced serum-induced retinal microvascular endothelial cell proliferation. Additionally, in a rat model of oxygen-induced retinopathy, retinal endoglin was significantly increased at 14(2), 14(3), 14(4) and 14(6) compared with retinal levels in control rats. At 14(2), immunohistochemical analysis demonstrated that endoglin was elevated in newly developed vessels at the peripheral extent of major veins, precisely where NV is expected to develop in OIR rats. Neutralizing anti-CD105 reduced retinal NV in OIR rats. CONCLUSIONS: Our data support other studies showing that reduction of endoglin expression inhibits retinal NV. Our findings demonstrate that retinal endoglin immunolocalization overlaps with nascent neovascular structures in OIR rats. Therefore, endoglin may serve as a useful predictor of incipient neovascular disease.
Assuntos
Animais Recém-Nascidos , Células Endoteliais/patologia , Peptídeos e Proteínas de Sinalização Intracelular/biossíntese , Neovascularização Patológica/metabolismo , Neovascularização Retiniana/metabolismo , Vasos Retinianos/metabolismo , Animais , Western Blotting , Proliferação de Células , Células Cultivadas , Modelos Animais de Doenças , Progressão da Doença , Endoglina , Células Endoteliais/metabolismo , Ensaio de Imunoadsorção Enzimática , Humanos , Imuno-Histoquímica , Recém-Nascido , Neovascularização Patológica/patologia , Ratos , Neovascularização Retiniana/patologia , Vasos Retinianos/patologiaRESUMO
PURPOSE: The purpose of this study was to investigate the role(s) of cytochrome P450 epoxygenases (CYPs) and their products, the epoxyeicosatrienoic acids (EETs), in hypoxia-induced VEGF production and pathologic retinal angiogenesis. METHODS: Human retinal astrocytes, Müller cells, and retinal microvascular endothelial cells (HRMEC) were exposed to hypoxia, and relative CYP2C expression was measured by RT-PCR. Astrocyte and Müller cell VEGF production was measured by ELISA after exposure to hypoxia and treatment with the general CYP inhibitor, SKF-525a. Human retinal microvascular endothelial cells were treated with the CYP product, 11,12-epoxyeicosatrienoic acid [EET], or SKF-525a in the presence or absence of VEGF. Proliferation of HRMEC and tube formation were assayed. Oxygen-induced retinopathy (OIR) was induced in newborn rats. Retinal CYP2C11 and CYP2C23 expression were measured by RT-PCR. The OIR rats received SKF-525a by intravitreal injection and preretinal neovascularization (NV) was quantified. Retinal VEGF protein levels were measured by ELISA. RESULTS: Human retinal astrocytes were the only cells to exhibit significant induction of CYP2C8 and CYP2C9 mRNA expression by hypoxia. Astrocytes, but not Müller cells, exhibited reduced hypoxia-induced VEGF production when treated with SKF-525a. 11,12-EET induced HRMEC proliferation and tube formation, and SKF-525a inhibited VEGF-induced proliferation. Oxygen-induced retinopathy induced expression of CYP2C23, but had no effect on CYP2C11. SKF-525a inhibited retinal NV and reduced retinal VEGF levels in OIR rats. CONCLUSIONS: The CYP-derived 11,12-EET may exhibit a proangiogenic biological function in the retina following stimulation by hypoxia in astrocytes. Inhibition of CYP may provide a rational therapy against retinal NV, because it can reduce VEGF production and VEGF-induced angiogenic responses in endothelial cells.
Assuntos
Sistema Enzimático do Citocromo P-450/genética , Regulação da Expressão Gênica no Desenvolvimento , RNA Mensageiro/genética , Retina/patologia , Neovascularização Retiniana/genética , Animais , Animais Recém-Nascidos , Hidrocarboneto de Aril Hidroxilases/biossíntese , Hidrocarboneto de Aril Hidroxilases/genética , Astrócitos/metabolismo , Astrócitos/patologia , Proliferação de Células , Células Cultivadas , Citocromo P-450 CYP2C8 , Citocromo P-450 CYP2C9 , Citocromo P-450 CYP2J2 , Sistema Enzimático do Citocromo P-450/biossíntese , Modelos Animais de Doenças , Endotélio Vascular/metabolismo , Endotélio Vascular/patologia , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Microcirculação , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase em Tempo Real , Retina/metabolismo , Neovascularização Retiniana/metabolismo , Neovascularização Retiniana/patologia , Fator A de Crescimento do Endotélio Vascular/biossínteseRESUMO
The purpose of this study was to investigate the hypoxia-induced Vegf120, Vegf164 and Vegf188 mRNA expression profiles in rat Müller cells (MC), astrocytes, retinal pigmented epithelial cells (RPE) and retinal microvascular endothelial cells (RMEC) and correlate these findings to VEGF secreted protein. Cultured cells were exposed to normoxia or hypoxia. Total RNA was isolated from cell lysates and Vegf splice variant mRNA copy numbers were assayed by a validated qRT-PCR external calibration curve method. mRNA copy numbers were normalized to input total RNA. Conditioned medium was collected from cells and assayed for total VEGF protein by ELISA. Hypoxia increased total Vegf mRNA and secreted protein in all the retinal cell types, with the highest levels observed in MC and astrocytes ranking second. Total Vegf mRNA levels in hypoxic RPE and RMEC were comparable; however, the greatest hypoxic induction of each Vegf splice variant mRNA was observed in RMEC. RPE and RMEC ranked 3rd and 4th respectively, in terms of secreted total VEGF protein in hypoxia. The Vegf120, Vegf164 and Vegf188 mRNA splice variants were all increased in hypoxic cells compared to normoxic controls. In normoxia, the relative Vegf splice variant mRNA levels ranked from highest to lowest for each cell type were Vegf164 > Vegf120 > Vegf188. Hypoxic induction did not alter this ranking, although it did favor an increased stoichiometry of Vegf164 mRNA over the other two splice variants. MC and astrocytes are likely to be the major sources of total Vegf, Vegf164 splice variant mRNAs, and VEGF protein in retinal hypoxia.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Hipóxia/genética , RNA Mensageiro/genética , Retina/metabolismo , Doenças Retinianas/genética , Fator A de Crescimento do Endotélio Vascular/genética , Animais , Animais Recém-Nascidos , Células Cultivadas , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Hipóxia/metabolismo , Hipóxia/patologia , Isoformas de Proteínas , Ratos , Ratos Long-Evans , Reação em Cadeia da Polimerase em Tempo Real , Retina/patologia , Doenças Retinianas/metabolismo , Doenças Retinianas/patologia , Fator A de Crescimento do Endotélio Vascular/biossínteseRESUMO
PURPOSE: To develop new therapies against ocular neovascularization (NV), we tested the effect of peroxisome proliferator-activated receptor-ß/δ (PPAR-ß/δ) agonism and antagonism on angiogenic behaviors and in human retinal microvascular endothelial cells (HRMEC) and on preretinal NV in rat oxygen-induced retinopathy (OIR). METHODS: HRMECs were treated with the PPAR-ß/δ agonist GW0742 and the antagonist GSK0660. Messenger RNA levels of a PPAR-ß/δ target gene, angiopoietin-like-4 (angptl4) were assayed by qRT-PCR. HRMEC proliferation and tube formation were assayed according to standard protocols. OIR was induced in newborn rats by exposing them to alternating 24-hour episodes of 50% and 10% oxygen for 14 days. OIR rats were treated with GW0742 or GSK0660. Angptl4 protein levels were assessed by ELISA and preretinal NV was quantified by adenosine diphosphatase staining. RESULTS: GW0742 significantly increased angptl4 mRNA, and GSK0660 significantly decreased angptl4 mRNA. GW0742 had no effect on HRMEC proliferation, but caused a significant and dose-responsive increase in tube formation. GSK0660 significantly reduced serum-induced HRMEC proliferation and tube formation in a dose-dependent manner. Intravitreal injection of GW0742 significantly increased total retinal Angptl4 protein, but intravitreal injection of GSK0660 had no effect. Intravitreal injection of GW0742 significantly increased retinal NV, as did GW0742 administered by oral gavage. Conversely, both intravitreal injection and intraperitoneal injection of GSK0660 significantly reduced retinal NV. CONCLUSIONS: PPAR-ß/δ activation exacerbates, and its inhibition reduces, preretinal NV. PPAR-ß/δ may regulate preretinal NV through a prodifferentiation/maturation mechanism that depends on Angptl4. Pharmacologic inhibition of PPAR-ß/δ may provide a rational basis for therapeutic targeting of ocular NV.