RESUMEN
Exosomes are nanosized vesicles that have been reported as cargo-delivering vehicles between cells. Müller cells play a crucial role in the pathogenesis of diabetic retinopathy (DR). Activated Müller cells in the diabetic retina mediate disruption of barrier integrity and neovascularization. Endothelial cells constitute the inner blood-retinal barrier (BRB). Herein, we aim to evaluate the effect of Müller cell-derived exosomes on endothelial cell viability and barrier function under normal and hyperglycemic conditions. Müller cell-derived exosomes were isolated and characterized using Western blotting, nanoparticle tracking, and electron microscopy. The uptake of Müller cells-derived exosomes by the human retinal endothelial cells (HRECs) was monitored by labeling exosomes with PKH67. Endothelial cell vitality after treatment by exosomes under normo- and hypoglycemic conditions was checked by MTT assay and Western blot for apoptotic proteins. The barrier function of HRECs was evaluated by analysis of ZO-1 and transcellular electrical resistance (TER) using ECIS. Additionally, intracellular Ca+2 in HRECs was assessed by spectrofluorimetry. Analysis of the isolated exosomes showed a non-significant change in the number of exosomes isolated from both normal and hyperglycemic condition media, however, the average size of exosomes isolated from the hyperglycemic group showed a significant rise when compared to that of the normoglycemic group. Müller cells derived exosomes from hyperglycemic condition media markedly reduced HRECs cell count, increased caspase-3 and Annexin V, decreased ZO-1 levels and TER, and increased intracellular Ca+ when compared to other groups. However, treatment of HRECs under hyperglycemia with normo-glycemic Müller cells-derived exosomes significantly decreased cell death, preserved cellular integrity and barrier function, and reduced intracellular Ca+2. Collectively, Müller cell-derived exosomes play a remarkable role in the pathological changes associated with hyperglycemia-induced inner barrier dysfunction in DR. Further in vivo research will help in understanding the role of exosomes as therapeutic targets and/or delivery systems for DR.
Asunto(s)
Apoptosis , Barrera Hematorretinal , Supervivencia Celular , Retinopatía Diabética , Células Endoteliales , Células Ependimogliales , Exosomas , Exosomas/metabolismo , Retinopatía Diabética/metabolismo , Retinopatía Diabética/patología , Retinopatía Diabética/fisiopatología , Células Ependimogliales/metabolismo , Células Ependimogliales/patología , Humanos , Células Endoteliales/metabolismo , Células Endoteliales/patología , Barrera Hematorretinal/metabolismo , Barrera Hematorretinal/patología , Células Cultivadas , Proteína de la Zonula Occludens-1/metabolismo , Permeabilidad Capilar , Señalización del Calcio , Línea Celular , Vasos Retinianos/metabolismo , Vasos Retinianos/patología , Vasos Retinianos/fisiopatologíaRESUMEN
Diabetic retinopathy (DR) is a neurovascular complication of diabetes, driven by an intricate network of cellular and molecular mechanisms. This study sought to explore the mechanisms by investigating the role of 12-hydroxyeicosatetraenoic acid (12-HETE), its receptor GPR31, and microRNA (miR-29) in the context of DR, specifically focusing on their impact on Müller glial cells. We found that 12-HETE activates Müller cells (MCs), elevates glutamate production, and induces inflammatory and oxidative responses, all of which are instrumental in DR progression. The expression of GPR31, the receptor for 12-HETE, was prominently found in the retina, especially in MCs and retinal ganglion cells, and was upregulated in diabetes. Interestingly, miR29 showed potential as a protective agent, mitigating the harmful effects of 12-HETE by attenuating inflammation and oxidative stress, and restoring the expression of pigment epithelium-derived factor (PEDF). Our results underline the central role of 12-HETE in DR progression through activation of a neurovascular toxic pathway in MCs and illuminate the protective capabilities of miR-29, highlighting both as promising therapeutic targets for the management of DR.
Asunto(s)
Retinopatía Diabética , MicroARNs , Ácido 12-Hidroxi-5,8,10,14-Eicosatetraenoico/metabolismo , Retinopatía Diabética/genética , Retinopatía Diabética/metabolismo , Células Ependimogliales , MicroARNs/genética , MicroARNs/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Retina/metabolismoRESUMEN
BACKGROUND: Cognitive decline is one of the aging health problems that strongly affects daily functioning and quality of life of older adults and threatens their independence. The aim of this study was to assess the prevalence and pattern of cognitive impairment (CI) among community-dwelling elderly in Egypt and the contribution of socioeconomic status to inequality in cognitive impairment. METHODS: A cross-sectional study involved 470 community-dwelling elderly aged 60 years or older living in Kafr El-Sheikh Governorate, Egypt. Subjects were recruited from home visits, geriatric clubs, and outpatient clinics. The Montreal Cognitive Assessment tools (MoCA & MoCA-B) were used to assess the prevalence of cognitive impairment, Hachinski ischemic score (HIS) to investigate the type of cognitive impairment, Ain Shams Cognitive Assessment (ASCA) tool to assess the pattern of specific cognitive domain affection, and an Egyptian socioeconomic status (SES) scale to classify the SES of the study participants. RESULTS: The prevalence of cognitive impairment was 50.2% distributed as 37.7% for mild cognitive impairment (MCI) and 12.5% for dementia. The most common type of cognitive impairment was the degenerative type (47.9%). Pattern of specific domain affection among cognitively impaired subjects ranged from 94% for visuospatial function to 12.7% for abstraction. Cognitive impairment was significantly higher with increasing age, female sex, marital status (single or widow), low education, higher number of comorbidities, and positive family history of cognitive impairment (p < 0.001). Also, cognitive impairment was concentrated mainly among participants with low socioeconomic score (p < 0.001). CONCLUSION: In Egypt, cognitive impairment is significantly prevalent and concentrated among those who are in low socioeconomic status. Patients with mild CI were more than those with dementia, and the most common type of CI was the degenerative type. Increasing educational level of low SES population and improving their access to healthcare services are highly recommended to improve the inequity of cognitive impairment.
RESUMEN
Age-related macular degeneration (AMD) is a major cause of blindness. Recent studies have reported impaired glycolysis in AMD patients with a high lactate/pyruvate ratio. Elevated homocysteine (Hcy) (Hyperhomocysteinemia, HHcy) was observed in several clinical studies, reporting an association between HHcy and AMD. We established the effect of HHcy on barrier function, retinal pigment epithelium (RPE) structure, and induced choroidal neovascularization (CNV) in mice. We hypothesize that HHcy contributes to AMD by inducing a metabolic switch in the mitochondria, in which cells predominantly produce energy by the high rate of glycolysis, or "Warburg", effect. Increased glycolysis results in an increased production of lactate, cellular acidity, activation of angiogenesis, RPE barrier dysfunction, and CNV. Evaluation of cellular energy production under HHcy was assessed by seahorse analysis, immunofluorescence, and western blot experiments. The seahorse analysis evaluated the extracellular acidification rate (ECAR) as indicative of glycolysis. HHcy showed a significant increase in ECAR both in vivo using (Cystathionine ß-synthase) cbs+/- and cbs-/- mice retinas and in vitro (Hcy-treated ARPE-19) compared to wild-type mice and RPE cells. Moreover, HHcy up-regulated glycolytic enzyme (Glucose transporter-1 (GlUT-1), lactate dehydrogenase (LDH), and hexokinase 1 (HK1)) in Hcy-treated ARPE-19 and primary RPE cells isolated from cbs+/+, cbs+/-, and cbs-/- mice retinas. Inhibition of GLUT-1 or blocking of N-methyl-D-aspartate receptors (NMDAR) reduced glycolysis in Hcy-treated RPE and improved albumin leakage and CNV induction in Hcy-injected mice eyes. The current study suggests that HHcy causes a metabolic switch in the RPE cells from mitochondrial respiration to glycolysis during AMD and confirms the involvement of NMDAR in this process. Therefore, targeting Glycolysis or NMDAR could be a novel therapeutic target for AMD.
Asunto(s)
Neovascularización Coroidal , Hiperhomocisteinemia , Degeneración Macular , Ratones , Animales , Células Cultivadas , Degeneración Macular/metabolismo , Epitelio Pigmentado de la Retina/metabolismo , Hiperhomocisteinemia/metabolismo , Neovascularización Coroidal/metabolismo , Cistationina betasintasa/metabolismo , Homocisteína/metabolismoRESUMEN
Hyperhomocysteinemia (HHcy) is remarkably common among the aging population. The relation between HHcy and the development of neurodegenerative diseases, such as Alzheimer's disease (AD) and eye diseases, and age-related macular degeneration (AMD) and diabetic retinopathy (DR) in elderly people, has been established. Disruption of the blood barrier function of the brain and retina is one of the most important underlying mechanisms associated with HHcy-induced neurodegenerative and retinal disorders. Impairment of the barrier function triggers inflammatory events that worsen disease pathology. Studies have shown that AD patients also suffer from visual impairments. As an extension of the central nervous system, the retina has been suggested as a prominent site of AD pathology. This review highlights inflammation as a possible underlying mechanism of HHcy-induced barrier dysfunction and neurovascular injury in aging diseases accompanied by HHcy, focusing on AD.
Asunto(s)
Enfermedades del Sistema Nervioso Central/patología , Homocisteína/metabolismo , Hiperhomocisteinemia/patología , Inflamación/fisiopatología , Factores de Edad , Animales , Enfermedades del Sistema Nervioso Central/etiología , Enfermedades del Sistema Nervioso Central/metabolismo , Humanos , Hiperhomocisteinemia/etiología , Hiperhomocisteinemia/metabolismoRESUMEN
Age-related macular degeneration (AMD) is a leading cause of vision loss. Elevated homocysteine (Hcy) (Hyperhomocysteinemia) (HHcy) has been reported in AMD. We previously reported that HHcy induces AMD-like features. This study suggests that N-Methyl-d-aspartate receptor (NMDAR) activation in the retinal pigment epithelium (RPE) is a mechanism for HHcy-induced AMD. Serum Hcy and cystathionine-ß-synthase (CBS) were assessed by ELISA. The involvement of NMDAR in Hcy-induced AMD features was evaluated (1) in vitro using ARPE-19 cells, primary RPE isolated from HHcy mice (CBS), and mouse choroidal endothelial cells (MCEC); (2) in vivo using wild-type mice and mice deficient in RPE NMDAR (NMDARR-/-) with/without Hcy injection. Isolectin-B4, Ki67, HIF-1α, VEGF, NMDAR1, and albumin were assessed by immunofluorescence (IF), Western blot (WB), Optical coherence tomography (OCT), and fluorescein angiography (FA) to evaluate retinal structure, fluorescein leakage, and choroidal neovascularization (CNV). A neovascular AMD patient's serum showed a significant increase in Hcy and a decrease in CBS. Hcy significantly increased HIF-1α, VEGF, and NMDAR in RPE cells, and Ki67 in MCEC. Hcy-injected WT mice showed disrupted retina and CNV. Knocking down RPE NMDAR improved retinal structure and CNV. Our findings underscore the role of RPE NMDAR in Hcy-induced AMD features; thus, NMDAR inhibition could serve as a promising therapeutic target for AMD.
Asunto(s)
Homocisteína/efectos adversos , Homocisteína/sangre , Degeneración Macular/metabolismo , Receptores de N-Metil-D-Aspartato/genética , Receptores de N-Metil-D-Aspartato/metabolismo , Anciano , Anciano de 80 o más Años , Animales , Línea Celular , Neovascularización Coroidal/etiología , Cistationina betasintasa/sangre , Modelos Animales de Enfermedad , Células Endoteliales/metabolismo , Femenino , Humanos , Hiperhomocisteinemia/complicaciones , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Degeneración Macular/inducido químicamente , Degeneración Macular/diagnóstico por imagen , Degeneración Macular/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Persona de Mediana Edad , Neovascularización Patológica/etiología , Cultivo Primario de Células , Epitelio Pigmentado de la Retina/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Clinical studies have suggested that the renin-angiotensin system (RAS) may be a promising therapeutic target in treating diabetic retinopathy (DR). While AT1 receptor blockade decreased the incidence of DR in the DIRECT trial, it did not reduce the DR progression. Lack of understanding of the molecular mechanism of retinal microvascular damage induced by RAS is a critical barrier to the use of RAS blockade in preventing or treating DR. The purpose of this study is to investigate the interaction between soluble epoxide hydrolase (sEH) and the AT1 receptor in Angiotensin II (Ang II)- and diabetes-induced retinal microvascular damage. We demonstrate that Ang II increases retinal sEH levels, which is blunted by an AT1 blocker; administration of 11,12-epoxyeicosatrienoic acid (EET) exacerbates intravitreal Ang II-induced retinal albumin leakage; while sEH knockout (KO) and blockade reduce Ang II-induced retinal vascular remodeling, sEH KO causes retinal vascular leakage in Ang II-sEH KO mice; and sEH KO potentiates diabetes-induced retinal damage via promoting retinal vascular endothelial growth factor (VEGF) but reducing expression of tight junction proteins (ZO-1 and occludin). Our studies hold the promise of providing a new strategy, the use of combined EETs blockade with AT1 blocker, to prevent or reduce DR.
Asunto(s)
Angiotensina II/metabolismo , Diabetes Mellitus Experimental/patología , Epóxido Hidrolasas/metabolismo , Microvasos/patología , Receptor de Angiotensina Tipo 1/metabolismo , Sistema Renina-Angiotensina , Retina/patología , Animales , Diabetes Mellitus Experimental/metabolismo , Modelos Animales de Enfermedad , Epóxido Hidrolasas/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microvasos/metabolismo , Retina/metabolismoRESUMEN
The potential therapeutic effects of agonistic analogs of growth hormone-releasing hormone (GHRH) and their mechanism of action were investigated in diabetic retinopathy (DR). Streptozotocin-induced diabetic rats (STZ-rats) were treated with 15 µg/kg GHRH agonist, MR-409, or GHRH antagonist, MIA-602. At the end of treatment, morphological and biochemical analyses assessed the effects of these compounds on retinal neurovascular injury induced by hyperglycemia. The expression levels of GHRH and its receptor (GHRH-R) measured by qPCR and Western blotting were significantly down-regulated in retinas of STZ-rats and in human diabetic retinas (postmortem) compared with their respective controls. Treatment of STZ-rats with the GHRH agonist, MR-409, prevented retinal morphological alteration induced by hyperglycemia, particularly preserving survival of retinal ganglion cells. The reverse, using the GHRH antagonist, MIA-602, resulted in worsening of retinal morphology and a significant alteration of the outer retinal layer. Explaining these results, we have found that MR-409 exerted antioxidant and anti-inflammatory effects in retinas of the treated rats, as shown by up-regulation of NRF-2-dependent gene expression and down-regulation of proinflammatory cytokines and adhesion molecules. MR-409 also significantly down-regulated the expression of vascular endothelial growth factor while increasing that of pigment epithelium-derived factor in diabetic retinas. These effects correlated with decreased vascular permeability. In summary, our findings suggest a neurovascular protective effect of GHRH analogs during the early stage of diabetic retinopathy through their antioxidant and anti-inflammatory properties.
Asunto(s)
Antiinflamatorios/farmacología , Retinopatía Diabética/tratamiento farmacológico , Hormona Liberadora de Hormona del Crecimiento/agonistas , Sermorelina/análogos & derivados , Animales , Antiinflamatorios/uso terapéutico , Moléculas de Adhesión Celular/genética , Moléculas de Adhesión Celular/metabolismo , Citocinas/genética , Citocinas/metabolismo , Retinopatía Diabética/metabolismo , Factor de Transcripción de la Proteína de Unión a GA/genética , Factor de Transcripción de la Proteína de Unión a GA/metabolismo , Hormona Liberadora de Hormona del Crecimiento/antagonistas & inhibidores , Humanos , Masculino , Ratas , Ratas Sprague-Dawley , Retina/efectos de los fármacos , Retina/metabolismo , Sermorelina/farmacología , Factor A de Crecimiento Endotelial Vascular/genética , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
AIMS: Our previous studies have established a role for 12/15-lipoxygenase (LO) in mediating the inflammatory response in diabetic retinopathy (DR). However, the extent at which the local or systemic induction of 12/15-LO activity involved is unclear. Thus, the current study aimed to characterize the relative contribution of retinal endothelial versus monocytic/macrophagic 12/15-LO to inflammatory responses in DR. MATERIALS & METHODS: We first generated a clustered heat map for circulating bioactive lipid metabolites in the plasma of streptozotocin (STZ)-induced diabetic mice using liquid chromatography coupled with mass-spectrometry (LC-MS) to evaluate changes in circulating 12/15-LO activity. This was followed by comparing the in vitro mouse endothelium-leukocytes interaction between leukocytes isolated from 12/15-LO knockout (KO) versus those isolated from wild type (WT) mice using the myeloperoxidase (MPO) assay. Finally, we examined the effects of knocking down or inhibiting endothelial 12/15-LO on diabetes-induced endothelial cell activation and ICAM-1 expression. RESULTS: Analysis of plasma bioactive lipids' heat map revealed that the activity of circulating 12/15-LO was not altered by diabetes as evident by no significant changes in the plasma levels of major metabolites derived from 12/15-lipoxygenation of different PUFAs, including linoleic acid (13-HODE), arachidonic acid (12- and 15- HETEs), eicosapentaenoic acid (12- and 15- HEPEs), or docosahexaenoic acid (17-HDoHE). Moreover, leukocytes from 12/15-LO KO mice displayed a similar increase in adhesion to high glucose (HG)-activated endothelial cells as do leukocytes from WT mice. Furthermore, abundant proteins of 12-LO and 15-LO were detected in human retinal endothelial cells (HRECs), while it was undetected (15-LO) or hardly detectable (12-LO) in human monocyte-like U937 cells. Inhibition or knock down of endothelial 12/15-LO in HRECs blocked HG-induced expression of ICAM-1, a well-known identified important molecule for leukocyte adhesion in DR. CONCLUSION: Our data support that endothelial, rather than monocytic/macrophagic, 12/15-LO has a critical role in hyperglycemia-induced ICAM-1 expression, leukocyte adhesion, and subsequent local retinal barrier dysfunction. This may facilitate the development of more precisely targeted treatment strategies for DR.
Asunto(s)
Araquidonato 12-Lipooxigenasa/metabolismo , Araquidonato 15-Lipooxigenasa/metabolismo , Retinopatía Diabética/enzimología , Células Endoteliales/enzimología , Leucostasis/enzimología , Macrófagos/enzimología , Monocitos/enzimología , Retina/enzimología , Animales , Araquidonato 12-Lipooxigenasa/genética , Araquidonato 15-Lipooxigenasa/genética , Adhesión Celular/genética , Retinopatía Diabética/genética , Retinopatía Diabética/patología , Células Endoteliales/patología , Humanos , Molécula 1 de Adhesión Intercelular/biosíntesis , Molécula 1 de Adhesión Intercelular/genética , Leucostasis/genética , Leucostasis/patología , Macrófagos/patología , Ratones , Ratones Noqueados , Monocitos/patología , Retina/patología , Células U937RESUMEN
We recently demonstrated that 12/15-lipoxygenase (LOX) derived metabolites, hydroxyeicosatetraenoic acids (HETEs), contribute to diabetic retinopathy (DR) via NADPH oxidase (NOX) and disruption of the balance in retinal levels of the vascular endothelial growth factor (VEGF) and pigment epithelium-derived factor (PEDF). Here, we test whether PEDF ameliorates retinal vascular injury induced by HETEs and the underlying mechanisms. Furthermore, we pursue the causal relationship between LOX-NOX system and regulation of PEDF expression during DR. For these purposes, we used an experimental eye model in which normal mice were injected intravitreally with 12-HETE with/without PEDF. Thereafter, fluorescein angiography (FA) was used to evaluate the vascular leakage, followed by optical coherence tomography (OCT) to assess the presence of angiogenesis. FA and OCT reported an increased vascular leakage and pre-retinal neovascularization, respectively, in response to 12-HETE that were not observed in the PEDF-treated group. Moreover, PEDF significantly attenuated the increased levels of vascular cell and intercellular adhesion molecules, VCAM-1 and ICAM-1, elicited by 12-HETE injection. Accordingly, the direct relationship between HETEs and PEDF has been explored through in-vitro studies using Müller cells (rMCs) and human retinal endothelial cells (HRECs). The results showed that 12- and 15-HETEs triggered the secretion of TNF-α and IL-6, as well as activation of NFκB in rMCs and significantly increased permeability and reduced zonula occludens protein-1 (ZO-1) immunoreactivity in HRECs. All these effects were prevented in PEDF-treated cells. Furthermore, interest in PEDF regulation during DR has been expanded to include NOX system. Retinal PEDF was significantly restored in diabetic mice treated with NOX inhibitor, apocynin, or lacking NOX2 up to 80% of the control level. Collectively, our findings suggest that interfering with LOX-NOX signaling opens up a new direction for treating DR by restoring endogenous PEDF that carries out multilevel vascular protective functions.
Asunto(s)
Ácido 12-Hidroxi-5,8,10,14-Eicosatetraenoico/antagonistas & inhibidores , Retinopatía Diabética/tratamiento farmacológico , Proteínas del Ojo/farmacología , Ácidos Hidroxieicosatetraenoicos/antagonistas & inhibidores , Factores de Crecimiento Nervioso/farmacología , Retina/efectos de los fármacos , Neovascularización Retiniana/tratamiento farmacológico , Serpinas/farmacología , Ácido 12-Hidroxi-5,8,10,14-Eicosatetraenoico/farmacología , Acetofenonas/farmacología , Animales , Araquidonato 12-Lipooxigenasa/genética , Araquidonato 12-Lipooxigenasa/metabolismo , Araquidonato 15-Lipooxigenasa/genética , Araquidonato 15-Lipooxigenasa/metabolismo , Células Cultivadas , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/patología , Retinopatía Diabética/genética , Retinopatía Diabética/metabolismo , Retinopatía Diabética/patología , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Células Endoteliales/patología , Células Ependimogliales/efectos de los fármacos , Células Ependimogliales/metabolismo , Células Ependimogliales/patología , Regulación de la Expresión Génica , Humanos , Ácidos Hidroxieicosatetraenoicos/farmacología , Molécula 1 de Adhesión Intercelular/genética , Molécula 1 de Adhesión Intercelular/metabolismo , Interleucina-6/genética , Interleucina-6/metabolismo , Inyecciones Intravítreas , Glicoproteínas de Membrana/antagonistas & inhibidores , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Ratones , Ratones Noqueados , NADPH Oxidasa 2 , NADPH Oxidasas/antagonistas & inhibidores , NADPH Oxidasas/genética , NADPH Oxidasas/metabolismo , FN-kappa B/genética , FN-kappa B/metabolismo , Retina/metabolismo , Retina/patología , Neovascularización Retiniana/genética , Neovascularización Retiniana/metabolismo , Neovascularización Retiniana/patología , Transducción de Señal , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/metabolismo , Molécula 1 de Adhesión Celular Vascular/genética , Molécula 1 de Adhesión Celular Vascular/metabolismo , Proteína de la Zonula Occludens-1/genéticaRESUMEN
Mutations in crumb homologue 1 (CRB1) in humans are associated with Leber's congenital amaurosis (LCA) and retinitis pigmentosa (RP). There is no clear genotype-phenotype correlation for human CRB1 mutations in RP and LCA. The high variability in clinical features observed in CRB1 mutations suggests that environmental factors or genetic modifiers influence severity of CRB1 related retinopathies. Retinal degeneration 8 (rd8) is a spontaneous mutation in the Crb1 gene (Crb1(rdr/rd8)). Crb1(rdr/rd8) mice present with focal disruption in the outer retina manifesting as white spots on fundus examination. Mild retinal dysfunction with decreased b-wave amplitude has been reported in Crb1(rdr/rd8) mice at 18 months. Methylene tetrahydrofolate reductase (MTHFR) is a crucial enzyme of homocysteine metabolism. MTHFR mutations are prevalent in humans and are linked to a broad spectrum of disorders including cardiovascular and neurodegenerative diseases. We recently reported the retinal phenotype in Mthfr-deficient (Mthfr(+/-)) heterozygous mice. At 24 weeks the mice showed decreased RGC function, thinner nerve fiber layer, focal areas of vascular leakage and 20% fewer cells in the ganglion cell layer (GCL). Considering the variability in CRB1-related retinopathies and the high occurrence of human MTHFR mutations we evaluated whether Mthfr deficiency influences rd8 retinal phenotype. Mthfr heterozygous mice with rd8 mutations (Mthfr(+/-)(rd8/rd8)) and Crb(rd8/rd8) mice (Mthfr(+/+rd8/rd8)) mice were subjected to comprehensive retinal evaluation using ERG, fundoscopy, fluorescein angiography (FA), morphometric and retinal flat mount immunostaining analyses of isolectin-B4 at 8-54 wks. Assessment of retinal function revealed a significant decrease in the a-, b- and c-wave amplitudes in Mthfr(+/-)(rd8/rd8) mice at 52 wks. Fundoscopic evaluation demonstrated the presence of signature rd8 spots in Mthfr(+/+rd8/rd8) mice and an increase in the extent of these rd8 spots in Mthfr(+/-)(rd8/rd8) mice at 24 weeks and beyond. FA revealed marked vascular leakage, ischemia and vascular tortuosity in Mthfr(+/-)(rd8/rd8) mice at 24 and 52 weeks. Retinal dysplasia was observed in â¼14-33% Mthfr(+/-)(rd8/rd8) mice by morphometric analysis. This was accompanied by a â¼20% reduction in cells of the GCL of Mthfr(+/-)(rd8/rd8) mice at 24 and 52 weeks. Retinal flat mount immunostaining with isolectin-B4 showed neovascularization and loss of blood vessel integrity in Mthfr(+/-)(rd8/rd8) mice in contrast to mild vasculopathy in Mthfr(+/+rd8/rd8) mice. Taken together, our data support an earlier onset and worsened retinal phenotype when Mthfr and rd8 mutations coexist. Our study sets the stage for future studies to investigate the role of MTHFR deficiency in human CRB1 retinopathies.
Asunto(s)
Metilenotetrahidrofolato Reductasa (NADPH2)/metabolismo , Proteínas del Tejido Nervioso/genética , Retina/metabolismo , Degeneración Retiniana/metabolismo , Células Ganglionares de la Retina/metabolismo , Animales , ADN/genética , Análisis Mutacional de ADN , Modelos Animales de Enfermedad , Angiografía con Fluoresceína , Fondo de Ojo , Estudios de Asociación Genética , Ratones , Ratones Endogámicos C57BL , Mutación , Proteínas del Tejido Nervioso/metabolismo , Fenotipo , Retina/patología , Degeneración Retiniana/genética , Degeneración Retiniana/patología , Células Ganglionares de la Retina/patologíaRESUMEN
Retinal hyperpermeability and subsequent macular edema is a cardinal feature of early diabetic retinopathy (DR). Here, we investigated the role of bioactive lipid metabolites, in particular 12/15-lipoxygenase (LOX)-derived metabolites, in this process. LC/MS lipidomic screen of human retinal endothelial cells (HRECs) demonstrated that 15-HETE was the only significantly increased metabolite (2.4 ± 0.4-fold, P = 0.0004) by high glucose (30 mM) treatment. In the presence of arachidonic acid, additional eicosanoids generated by 12/15-LOX, including 12- and 11-HETEs, were significantly increased. Fluorescein angiography and retinal albumin leakage showed a significant decrease in retinal hyperpermeability in streptozotocin-induced diabetic mice lacking 12/15-LOX compared with diabetic WT mice. Our previous studies demonstrated the potential role of NADPH oxidase in mediating the permeability effect of 12- and 15-HETEs, therefore we tested the impact of intraocular injection of 12-HETE in mice lacking the catalytic subunit of NADPH oxidase (NOX2). The permeability effect of 12-HETE was significantly reduced in NOX2(-/-) mice compared with the WT mice. In vitro experiments also showed that 15-HETE induced HREC migration and tube formation in a NOX-dependent manner. Taken together our data suggest that 12/15-LOX is implicated in DR via a NOX-dependent mechanism.
Asunto(s)
Araquidonato 12-Lipooxigenasa/metabolismo , Araquidonato 15-Lipooxigenasa/metabolismo , Retinopatía Diabética/tratamiento farmacológico , Ácidos Hidroxieicosatetraenoicos/farmacología , Hiperglucemia/tratamiento farmacológico , Glicoproteínas de Membrana/metabolismo , NADPH Oxidasas/metabolismo , Animales , Araquidonato 12-Lipooxigenasa/genética , Araquidonato 15-Lipooxigenasa/genética , Retinopatía Diabética/enzimología , Retinopatía Diabética/genética , Retinopatía Diabética/patología , Humanos , Hiperglucemia/enzimología , Hiperglucemia/genética , Hiperglucemia/patología , Glicoproteínas de Membrana/genética , Ratones , Ratones Noqueados , NADPH Oxidasa 2 , NADPH Oxidasas/genéticaRESUMEN
The high-affinity sigma receptor 1 (σR1) ligand (+)-pentazocine ((+)-PTZ) affords profound retinal neuroprotection in vitro and in vivo by a yet-unknown mechanism. A common feature of retinal disease is Müller cell reactive gliosis, which includes cytokine release. Here, we investigated whether lipopolysaccharide (LPS) stimulates cytokine release by primary mouse Müller cells and whether (+)-PTZ alters release. Using a highly sensitive inflammatory antibody array we observed significant release of macrophage inflammatory proteins (MIP1γ, MIP2, MIP3α) and interleukin-12 (IL12 (p40/p70)) in LPS-treated cells compared to controls, and a significant decrease in secretion upon (+)-PTZ treatment. Müller cells from σR1 knockout mice demonstrated increased MIP1γ, MIP2, MIP3α and IL12 (p40/p70) secretion when exposed to LPS compared to LPS-stimulated WT cells. We investigated whether cytokine secretion was accompanied by cytosolic-to-nuclear NFκB translocation and whether endothelial cell adhesion/migration was altered by released cytokines. Cells exposed to LPS demonstrated increased NFκB nuclear location, which was reduced significantly in (+)-PTZ-treated cells. Media conditioned by LPS-stimulated-Müller cells induced leukocyte-endothelial cell adhesion and endothelial cell migration, which was attenuated by (+)-PTZ treatment. The findings suggest that release of certain inflammatory cytokines by Müller cells can be attenuated by σR1 ligands providing insights into the retinal neuroprotective role of this receptor.
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Citocinas/metabolismo , Células Ependimogliales/metabolismo , Inflamación/metabolismo , Fármacos Neuroprotectores/farmacología , Pentazocina/farmacología , Receptores sigma/metabolismo , Animales , Movimiento Celular , Ensayo de Inmunoadsorción Enzimática , Células Ependimogliales/efectos de los fármacos , Células Ependimogliales/inmunología , Inmunohistoquímica , Inflamación/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptores sigma/inmunología , Receptor Sigma-1RESUMEN
Mild to moderate hyperhomocysteinemia is prevalent in humans and is implicated in neurovascular diseases, including recently in certain retinal diseases. Herein, we used hyperhomocysteinemic mice deficient in the Cbs gene encoding cystathionine-ß-synthase (Cbs(+/-)) to evaluate retinal vascular integrity. The Cbs(+/+) (wild type) and Cbs(+/-) (heterozygous) mice (aged 16 to 52 weeks) were subjected to fluorescein angiography and optical coherence tomography to assess vasculature in vivo. Retinas harvested for cryosectioning or flat mount preparations were subjected to immunofluorescence microscopy to detect blood vessels (isolectin-B4), angiogenesis [anti-vascular endothelial growth factor (VEGF) and anti-CD105], gliosis [anti-glial fibrillary acidic protein (GFAP)], pericytes (anti-neural/glial antigen 2), blood-retinal barrier [anti-zonula occludens protein 1 (ZO-1) and anti-occludin], and hypoxia [anti-pimonidazole hydrochloride (Hypoxyprobe-1)]. Levels of VEGF, GFAP, ZO-1, and occludin were determined by immunoblotting. Results of these analyses showed a mild vascular phenotype in young mice, which progressed with age. Fluorescein angiography revealed progressive neovascularization and vascular leakage in Cbs(+/-) mice; optical coherence tomography confirmed new vessels in the vitreous by 1 year. Immunofluorescence microscopy demonstrated vascular patterns consistent with ischemia, including a capillary-free zone centrally and new vessels with capillary tufts midperipherally in older mice. This was associated with increased VEGF, CD105, and GFAP and decreased ZO-1/occludin levels in the Cbs(+/-) retinas. Retinal vein occlusion was observed in some Cbs(+/-) mouse retinas. We conclude that mild to moderate elevation of homocysteine in Cbs(+/-) mice is accompanied by progressive alterations in retinal vasculature characterized by ischemia, neovascularization, incompetent blood-retinal barrier, and vascular occlusion.
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Cistationina betasintasa/genética , Modelos Animales de Enfermedad , Hiperhomocisteinemia/patología , Vasos Retinianos/patología , Animales , Heterocigoto , Hiperhomocisteinemia/genética , Ratones , Ratones Mutantes , Microscopía Fluorescente , Reacción en Cadena en Tiempo Real de la Polimerasa , Neovascularización Retiniana/genética , Neovascularización Retiniana/patologíaRESUMEN
BACKGROUND: The pathogenesis of inflammatory bowel disease (IBD) and colorectal cancer (CRC) is thought to be related to immune response against gut microbiota. TLR4, IgA, and EpCAM have a role in intestinal local immune response and their altered expression related to both IBD and CRC. Lipopolysaccharide (LPS) is the main activator of TLR4. The objective of this study is to evaluate the possible role of intestinal microbiota in the pathogenesis of IBD and CRC through expression of TLR4, IgA and EpCAM. METHODS: One hundred five cases were divided into (Group 1/ Control: 10 sections of normal colonic mucosa, Group 2/CRC: 51 cases, Group 3/IBD: 44 cases). Immunohistochemistry for TLR4, IgA, and EpCAM was done. LPS was assessed in all groups. TLR4 gene and protein expression were assessed in colorectal cancer cell line by RT-PCR and immunocytochemistry. RESULTS: There was a significant correlation between TLR4 and tumor grade (P value 0.003 and 0.01 respectively). A significant correlation was found between IgA expression and T stage (P value 0.02) and between EpCAM expression and histologic type (P value 0.02). In comparison of CRC patients to controls; there was a statistically significant different expression of TLR4 positivity, IgA positivity and EpCAM (P value <0.001, 0.004, <0.001 respectively). Patients with CRC were compared to colitis patients and there was a statistically significant different expression of IgA positivity and EpCAM expression (P value <0.001). There was significant higher expression of TLR4 in CRC cell line than the fibroblast by both PCR and immunocytochemistry (P-value: 0.003 and 0.024 respectively). LPS level in CRC patients was significantly higher than the control and IBD groups (P values <0.001 and <0.001 respectively). CONCLUSION: TLR4, IgA, EpCAM expression in both CRC and IBD might be related to the pathogenic role of microbiota and could represent potential prevention modalities and therapeutic targets.
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Neoplasias Colorrectales , Enfermedades Inflamatorias del Intestino , Microbiota , Humanos , Neoplasias Colorrectales/patología , Receptor Toll-Like 4/genética , Lipopolisacáridos , Molécula de Adhesión Celular Epitelial/genética , Enfermedades Inflamatorias del Intestino/metabolismo , Inmunoglobulina ARESUMEN
Haemochromatosis is a genetic disorder of iron overload resulting from loss-of-function mutations in genes coding for the iron-regulatory proteins HFE (human leucocyte antigen-like protein involved in iron homoeostasis), transferrin receptor 2, ferroportin, hepcidin and HJV (haemojuvelin). Recent studies have established the expression of all of the five genes in the retina, indicating their importance in retinal iron homoeostasis. Previously, we demonstrated that HJV is expressed in RPE (retinal pigment epithelium), the outer and inner nuclear layers and the ganglion cell layer. In the present paper, we report on the consequences of Hjv deletion on the retina in mice. Hjv-/- mice at ≥18 months of age had increased iron accumulation in the retina with marked morphological damage compared with age-matched controls; these changes were not found in younger mice. The retinal phenotype in Hjv-/- mice included hyperplasia of RPE. We isolated RPE cells from wild-type and Hjv-/- mice and examined their growth patterns. Hjv-/- RPE cells were less senescent and exhibited a hyperproliferative phenotype. Hjv-/- RPE cells also showed up-regulation of Slc7a11 (solute carrier family 7 member 11 gene), which encodes the 'transporter proper' subunit xCT in the heterodimeric amino acid transporter xCT/4F2hc (cystine/glutamate exchanger). BMP6 (bone morphogenetic protein 6) could not induce hepcidin expression in Hjv-/- RPE cells, confirming that retinal cells require HJV for induction of hepcidin via BMP6 signalling. HJV is a glycosylphosphatidylinositol-anchored protein, and the membrane-associated HJV is necessary for BMP6-mediated activation of hepcidin promoter in RPE cells. Taken together, these results confirm the biological importance of HJV in the regulation of iron homoeostasis in the retina and in RPE.
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Proteínas Reguladoras del Hierro/metabolismo , Hierro/farmacología , Proteínas de la Membrana/fisiología , Retina/metabolismo , Degeneración Retiniana/inducido químicamente , Epitelio Pigmentado de la Retina/metabolismo , Envejecimiento/fisiología , Sistema de Transporte de Aminoácidos y+/biosíntesis , Animales , Péptidos Catiónicos Antimicrobianos/biosíntesis , Proteína Morfogenética Ósea 6/farmacología , Proteínas Ligadas a GPI , Proteína de la Hemocromatosis , Hepcidinas , Antígenos de Histocompatibilidad Clase I/genética , Hierro/metabolismo , Masculino , Proteínas de la Membrana/deficiencia , Proteínas de la Membrana/genética , Ratones , Ratones Noqueados , Degeneración Retiniana/metabolismo , Epitelio Pigmentado de la Retina/patologíaRESUMEN
Age-related macular degeneration (AMD) is a leading cause of blindness, and elucidating its underlying disease mechanisms is vital to the development of appropriate therapeutics. We identified differentially expressed genes (DEGs) and differentially spliced genes (DSGs) across the clinical stages of AMD in disease-affected tissue, the macular retina pigment epithelium (RPE)/choroid and the macular neural retina within the same eye. We utilized 27 deeply phenotyped donor eyes (recovered within a 6 h postmortem interval time) from Caucasian donors (60-94 years) using a standardized published protocol. Significant findings were then validated in an independent set of well-characterized donor eyes (n = 85). There was limited overlap between DEGs and DSGs, suggesting distinct mechanisms at play in AMD pathophysiology. A greater number of previously reported AMD loci overlapped with DSGs compared to DEGs between disease states, and no DEG overlap with previously reported loci was found in the macular retina between disease states. Additionally, we explored allele-specific expression (ASE) in coding regions of previously reported AMD risk loci, uncovering a significant imbalance in C3 rs2230199 and CFH rs1061170 in the macular RPE/choroid for normal eyes and intermediate AMD (iAMD), and for CFH rs1061147 in the macular RPE/choroid for normal eyes and iAMD, and separately neovascular AMD (NEO). Only significant DEGs/DSGs from the macular RPE/choroid were found to overlap between disease states. STAT1, validated between the iAMD vs. normal comparison, and AGTPBP1, BBS5, CERKL, FGFBP2, KIFC3, RORα, and ZNF292, validated between the NEO vs. normal comparison, revealed an intricate regulatory network with transcription factors and miRNAs identifying potential upstream and downstream regulators. Findings regarding the complement genes C3 and CFH suggest that coding variants at these loci may influence AMD development via an imbalance of gene expression in a tissue-specific manner. Our study provides crucial insights into the multifaceted genomic underpinnings of AMD (i.e., tissue-specific gene expression changes, potential splice variation, and allelic imbalance), which may open new avenues for AMD diagnostics and therapies specific to iAMD and NEO.
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D-Ala-D-Ala Carboxipeptidasa de Tipo Serina , Degeneración Macular Húmeda , Humanos , Alelos , Inhibidores de la Angiogénesis , Factor A de Crecimiento Endotelial Vascular , Agudeza Visual , Expresión Génica , Proteínas del Citoesqueleto , Proteínas de Unión a Fosfato , Proteínas Portadoras , Proteínas del Tejido Nervioso , Proteínas de Unión al GTPRESUMEN
Fumaric acid esters are used to treat psoriasis, an inflammatory skin disease characterized by keratinocyte proliferation. Inflammation and proliferation are hallmarks of retinal disease; hence, fumaric acid esters may have therapeutic value in retinal pathology. In diseased retinas, Müller glial cells (MCs) undergo reactive gliosis, a hyperproliferative state. MCs take up folate, a vitamin necessary for cell proliferation, via the proton-coupled folate transporter (PCFT). Here we examined the effect of monomethylfumarate (MMF), the active metabolite of fumaric acid esters, on expression and function of PCFT in MCs. Primary MCs, isolated from neonatal mouse retinas, were treated with MMF, and PCFT function was monitored by measuring uptake of radiolabeled methyltetrahydrofolate (MTF) at pH 5.5. Dose-response and time-course analyses were performed to identify optimal conditions for maximal effect. The influence of MMF treatment on kinetic parameters of PCFT was studied, and PCFT expression was analyzed at the mRNA and protein level. MTF uptake in MCs decreased by Ë50% following 18 h treatment with 1 mM MMF. This effect was specific to fumaric acid esters. MMF treatment decreased the maximal velocity of the transporter without altering substrate affinity. The decrease in PCFT function following MMF treatment was accompanied by attenuated PCFT expression. This is the first report that an antipsoriatic compound can regulate folate transport in MCs and may have potential for the treatment of reactive gliosis in retinal disease.
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Fármacos Dermatológicos/farmacología , Fumaratos/farmacología , Maleatos/farmacología , Neuroglía/efectos de los fármacos , Retina/citología , Análisis de Varianza , Animales , Animales Recién Nacidos , Antipsicóticos , Relación Dosis-Respuesta a Droga , Receptor 1 de Folato/genética , Receptor 1 de Folato/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Niacina/farmacología , Transportador de Folato Acoplado a Protón/genética , Transportador de Folato Acoplado a Protón/metabolismo , ARN Mensajero/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Receptores Nicotínicos/genética , Receptores Nicotínicos/metabolismo , Factores de Tiempo , Tritio/metabolismo , Vasodilatadores/farmacologíaRESUMEN
PURPOSE: Sigma receptor 1 (σR1) is a non-opioid transmembrane protein that may act as a molecular chaperone at the endoplasmic reticulum-mitochondrial membrane. Ligands for σR1, such as (+)-pentazocine [(+)-PTZ], confer marked retinal neuroprotection in vivo and in vitro. Recently we analyzed the retinal phenotype of mice lacking σR1 (σR1 KO) and observed normal retinal morphology and function in young mice (5-30 weeks) but diminished negative scotopic threshold responses (nSTRs), retinal ganglion cell (RGC) loss, and disruption of optic nerve axons consistent with inner retinal dysfunction by 1 year. These data led us to test the hypothesis that σR1 may be critical in forestalling chronic retinal stress; diabetes was used as the model of chronic stress. METHODS: To determine whether σR1 is required for (+)-PTZ neuroprotective effects, primary RGCs isolated from wild-type (WT) and σR1 KO mice were exposed to xanthine-xanthine oxidase (10 µM:2 mU/ml) to induce oxidative stress in the presence or absence of (+)-PTZ. Cell death was evaluated by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) analysis. To assess effects of chronic stress on RGC function, diabetes was induced in 3-week C57BL/6 (WT) and σR1 KO mice, using streptozotocin to yield four groups: WT nondiabetic (WT non-DB), WT diabetic (WT-DB), σR1 KO non-DB, and σR1 KO-DB. After 12 weeks of diabetes, when mice were 15-weeks old, intraocular pressure (IOP) was recorded, electrophysiologic testing was performed (including detection of nSTRs), and the number of RGCs was counted in retinal histological sections. RESULTS: In vitro studies showed that (+)-PTZ could not prevent oxidative stress-induced death of RGCs harvested from σR1 KO mice but afforded robust protection against death of RGCs harvested from WT mice. In the studies of chronic stress induced by diabetes, the IOP measured in the four mouse groups was within the normal range; however, there was a significant increase in the IOP of σR1 KO-DB mice (16 ± 0.5 mmHg) compared to the other groups tested (σR1 KO non-DB, WT non-DB, WT-DB: ~12 ± 0.6 mmHg). Regarding electrophysiologic testing, the nSTRs of σR1 KO non-DB mice were similar to WT non-DB mice at 15 weeks; however, they were significantly lower in σR1 KO-DB mice (5 ± 1 µV) compared to the other groups, including, notably, σR1 KO-nonDB (12±2 µV). As expected, the number of RGCs in σR1 KO non-DB mice was similar to WT non-DB mice at 15 weeks, but under chronic stress of diabetes there were fewer RGCs in retinas of σR1 KO-DB mice. CONCLUSIONS: This is the first report showing unequivocally that the neuroprotective effects of (+)-PTZ require σR1. σR1 KO mice show normal retinal structure and function at young ages; however, when subjected to the chronic stress of diabetes, there is an acceleration of retinal functional deficits in σR1 KO mice such that ganglion cell dysfunction is observed at a much earlier age than nondiabetic σR1 KO mice. The data support the hypothesis that σR1 plays a key role in modulating retinal stress and may be an important target for retinal disease.
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Envejecimiento , Diabetes Mellitus Experimental/genética , Retinopatía Diabética/genética , Receptores sigma/genética , Células Ganglionares de la Retina/metabolismo , Animales , Muerte Celular/efectos de los fármacos , Diabetes Mellitus Experimental/complicaciones , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/patología , Retinopatía Diabética/complicaciones , Retinopatía Diabética/metabolismo , Retinopatía Diabética/patología , Femenino , Eliminación de Gen , Etiquetado Corte-Fin in Situ , Presión Intraocular , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Fármacos Neuroprotectores/farmacología , Estrés Oxidativo , Pentazocina/farmacología , Cultivo Primario de Células , Receptores sigma/deficiencia , Células Ganglionares de la Retina/patología , Tonometría Ocular , Xantina Oxidasa/farmacología , Receptor Sigma-1RESUMEN
Homocysteine is an amino acid required for the metabolism of methionine. Excess homocysteine is implicated in cardiovascular and neurological disease and new data suggest a role in various retinopathies. Mice lacking cystathionine-beta-synthase (cbs(-/-)) have an excess of retinal homocysteine and develop anatomical abnormalities in multiple retinal layers, including photoreceptors and ganglion cells; heterozygous (cbs(+/-)) mice demonstrate ganglion cell loss and mitochondrial abnormalities in the optic nerve. The purpose of the present study was to determine whether elevated homocysteine, due to absent or diminished cbs, alters visual function. We examined cbs(-/-) (3 weeks) and cbs(+/-) mice (5, 10, 15, 30 weeks) and results were compared to those obtained from wild type (WT) littermates. Conventional dark- and light-adapted ERGs were recorded, along with dc-ERG to assess retinal pigment epithelial (RPE) function. The visual evoked potential (VEP) was used to assess transmission to the visual cortex. The amplitudes of the major ERG components were reduced in cbs(-/-) mice at age 3 weeks and VEPs were delayed markedly. These findings are consistent with the early retinal disruption observed anatomically in these mice. In comparison, at 3 weeks of age, responses of cbs(+/-) mice did not differ significantly from those of WT mice. Functional abnormalities were not observed in cbs(+/-) mice until 15 weeks of age, at which time amplitude reductions were noted for the ERG a- and b-wave and the light peak component, but not for other components generated by the RPE. VEP implicit times were delayed in cbs(+/-) mice at 15 and 30 weeks, while VEP amplitudes were unaffected. The later onset of functional defects in cbs(+/-) mice is consistent with a slow loss of ganglion cells reported previously in the heterozygous mutant. Light peak abnormalities indicate that RPE function is also compromised in older cbs(+/-) mice. The data suggest that severe elevations of homocysteine are associated with marked alterations of retinal function while modest homocysteine elevation is reflected in milder and delayed alterations of retinal function. The work lays the foundation to explore the role of homocysteine in retinal diseases such as glaucoma and optic neuropathy.