RESUMEN
BACKGROUND: Dietary-resistant starch is emerging as a potential therapeutic tool to limit the negative effects of diabetes on the kidneys. However, its metabolic and immunomodulatory effects have not yet been fully elucidated. METHODS: Six-week-old db/db mice were fed a diet containing 12.5% resistant starch or a control diet matched for equivalent regular starch for 10 weeks. db/m mice receiving the control diet were utilised as non-diabetic controls. Freshly collected kidneys were digested for flow cytometry analysis of immune cell populations. Kidney injury was determined by measuring albuminuria, histology, and immunohistochemistry. Portal vein plasma was collected for targeted analysis of microbially-derived metabolites. Intestinal histology and tight junction protein expression were assessed. RESULTS: Resistant starch limited the development of albuminuria in db/db mice. Diabetic db/db mice displayed a decline in portal vein plasma levels of acetate, propionate, and butyrate, which was increased with resistant starch supplementation. Diabetic db/db mice receiving resistant starch had a microbially-derived metabolite profile similar to that of non-diabetic db/m mice. The intestinal permeability markers lipopolysaccharide and lipopolysaccharide binding protein were increased in db/db mice consuming the control diet, which was not seen in db/db mice receiving resistant starch supplementation. Diabetes was associated with an increase in the kidney neutrophil population, neutrophil activation, number of C5aR1+ neutrophils, and urinary complement C5a excretion, all of which were reduced with resistant starch. These pro-inflammatory changes appear independent of fibrotic changes in the kidney. CONCLUSIONS: Resistant starch supplementation in diabetes promotes beneficial circulating microbially-derived metabolites and improves intestinal permeability, accompanied by a modulation in the inflammatory profile of the kidney including neutrophil infiltration, complement activation, and albuminuria. These findings indicate that resistant starch can regulate immune and inflammatory responses in the kidney and support the therapeutic potential of resistant starch supplementation in diabetes on kidney health.
Asunto(s)
Albuminuria , Nefropatías Diabéticas , Riñón , Infiltración Neutrófila , Animales , Ratones , Riñón/metabolismo , Masculino , Infiltración Neutrófila/efectos de los fármacos , Nefropatías Diabéticas/metabolismo , Nefropatías Diabéticas/dietoterapia , Almidón Resistente/farmacología , Microbioma Gastrointestinal/efectos de los fármacos , Almidón/farmacología , Diabetes Mellitus Experimental/metabolismo , Ratones Endogámicos C57BLRESUMEN
AIMS: Prevention of human hypertension is an important challenge and has been achieved in experimental models. Brief treatment with renin-angiotensin system (RAS) inhibitors permanently reduces the genetic hypertension of the spontaneously hypertensive rat (SHR). The kidney is involved in this fascinating phenomenon, but relevant changes in gene expression are unknown. METHODS AND RESULTS: In SHR, we studied the effect of treatment between 10 and 14 weeks of age with the angiotensin receptor blocker, losartan, or the angiotensin-converting enzyme inhibitor, perindopril [with controls for non-specific effects of lowering blood pressure (BP)], on differential RNA expression, DNA methylation, and renin immunolabelling in the kidney at 20 weeks of age. RNA sequencing revealed a six-fold increase in renin gene (Ren) expression during losartan treatment (P < 0.0001). Six weeks after losartan, arterial pressure remained lower (P = 0.006), yet kidney Ren showed reduced expression by 23% after losartan (P = 0.03) and by 43% after perindopril (P = 1.4 × 10-6) associated with increased DNA methylation (P = 0.04). Immunolabelling confirmed reduced cortical renin after earlier RAS blockade (P = 0.002). RNA sequencing identified differential expression of mRNAs, miRNAs, and lncRNAs with evidence of networking and co-regulation. These included 13 candidate genes (Grhl1, Ammecr1l, Hs6st1, Nfil3, Fam221a, Lmo4, Adamts1, Cish, Hif3a, Bcl6, Rad54l2, Adap1, Dok4), the miRNA miR-145-3p, and the lncRNA AC115371. Gene ontogeny analyses revealed that these networks were enriched with genes relevant to BP, RAS, and the kidneys. CONCLUSION: Early RAS inhibition in SHR resets genetic pathways and networks resulting in a legacy of reduced Ren expression and BP persisting for a minimum of 6 weeks.
Asunto(s)
Bloqueadores del Receptor Tipo 1 de Angiotensina II , Inhibidores de la Enzima Convertidora de Angiotensina , Antihipertensivos , Metilación de ADN , Modelos Animales de Enfermedad , Redes Reguladoras de Genes , Hipertensión , Riñón , Losartán , Perindopril , Ratas Endogámicas SHR , Sistema Renina-Angiotensina , Renina , Animales , Sistema Renina-Angiotensina/efectos de los fármacos , Sistema Renina-Angiotensina/genética , Riñón/metabolismo , Riñón/efectos de los fármacos , Losartán/farmacología , Hipertensión/fisiopatología , Hipertensión/genética , Hipertensión/tratamiento farmacológico , Hipertensión/metabolismo , Metilación de ADN/efectos de los fármacos , Masculino , Antihipertensivos/farmacología , Renina/genética , Renina/metabolismo , Inhibidores de la Enzima Convertidora de Angiotensina/farmacología , Bloqueadores del Receptor Tipo 1 de Angiotensina II/farmacología , Perindopril/farmacología , Factores de Tiempo , Epigénesis Genética/efectos de los fármacos , Regulación de la Expresión Génica , Presión Arterial/efectos de los fármacos , Transcriptoma , Ratas , Presión Sanguínea/efectos de los fármacos , Presión Sanguínea/genéticaRESUMEN
Retinal inflammation is a central feature of ocular neovascular diseases such as diabetic retinopathy and retinopathy of prematurity, but the contribution of neutrophils to this process is not fully understood. We studied oxygen-induced retinopathy (OIR) which develops in two phases, featuring hyperoxia-induced retinal vaso-obliteration in phase I, followed by retinal neovascularization in phase II. As neutrophils are acute responders to tissue damage, we evaluated whether neutrophil depletion with an anti-Ly6G mAb administered in phase I OIR influenced retinal inflammation and vascular injury. Neutrophils were measured in blood and spleen via flow cytometry, and myeloperoxidase, an indicator of neutrophil activity, was evaluated in the retina using Western blotting. Retinal vasculopathy was assessed by quantitating vaso-obliteration, neovascularization, vascular leakage, and VEGF levels. The inflammatory factors, TNF, MCP-1, and ICAM-1 were measured in retina. In the OIR controls, neutrophils were increased in the blood and spleen in phase I but not phase II OIR. In OIR, the anti-Ly6G mAb reduced neutrophils in the blood and spleen, and myeloperoxidase, inflammation, and vasculopathy in the retina. Our findings revealed that the early rise in neutrophils in OIR primes the retina for an inflammatory and angiogenic response that promotes severe damage to the retinal vasculature.
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Neovascularización Retiniana , Retinopatía de la Prematuridad , Animales , Ratones , Oxígeno/efectos adversos , Neutrófilos , Peroxidasa , Retinopatía de la Prematuridad/inducido químicamente , Factor A de Crecimiento Endotelial Vascular/fisiología , Animales Recién Nacidos , Retina , Neovascularización Patológica , Inflamación , Ratones Endogámicos C57BL , Modelos Animales de EnfermedadRESUMEN
Vision loss in diabetic retinopathy features damage to the blood-retinal barrier and neovascularization, with hypertension and the renin-angiotensin system (RAS) having causal roles. We evaluated if finerenone, a non-steroidal mineralocorticoid receptor (MR) antagonist, reduced vascular pathology and inflammation in diabetic and neovascular retinopathy. Diabetic and hypertensive transgenic (mRen-2)27 rats overexpressing the RAS received the MR antagonist finerenone (10 mg/kg/day, oral gavage) or the angiotensin-converting enzyme inhibitor perindopril (10 mg/kg/day, drinking water) for 12 weeks. As retinal neovascularization does not develop in diabetic rodents, finerenone (5 mg/kg/day, i.p.) was evaluated in murine oxygen-induced retinopathy (OIR). Retinal vasculopathy was assessed by measuring gliosis, vascular leakage, neovascularization, and VEGF. Inflammation was investigated by quantitating retinal microglia/macrophages, pro-inflammatory mediators, and anti-inflammatory regulatory T-cells (Tregs). In diabetes, both treatments reduced systolic blood pressure, gliosis, vascular leakage, and microglial/macrophage density, but only finerenone lowered VEGF, ICAM-1, and IL-1ß. In OIR, finerenone reduced neovascularization, vascular leakage, and microglial density, and increased Tregs in the blood, spleen, and retina. Our findings, in the context of the FIDELIO-DKD and FIGARO-DKD trials reporting the benefits of finerenone on renal and cardiovascular outcomes in diabetic kidney disease, indicate the potential of finerenone as an effective oral treatment for diabetic retinopathy.
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Diabetes Mellitus Tipo 2 , Nefropatías Diabéticas , Retinopatía Diabética , Lesiones del Sistema Vascular , Ratas , Animales , Ratones , Antagonistas de Receptores de Mineralocorticoides/farmacología , Antagonistas de Receptores de Mineralocorticoides/uso terapéutico , Retinopatía Diabética/tratamiento farmacológico , Retinopatía Diabética/complicaciones , Roedores , Gliosis/complicaciones , Factor A de Crecimiento Endotelial Vascular , Linfocitos T Reguladores , Naftiridinas/farmacología , Nefropatías Diabéticas/etiología , Neovascularización Patológica/complicaciones , Inflamación/complicaciones , Diabetes Mellitus Tipo 2/complicacionesRESUMEN
BACKGROUND: CD4+ (cluster of differentation) and CD8+ T cells are increased in the ocular fluids of patients with neovascular retinopathy, yet their role in the disease process is unknown. METHODS: We describe how CD8+ T cells migrate into the retina and contribute to pathological angiogenesis by releasing cytokines and cytotoxic factors. RESULTS: In oxygen-induced retinopathy, flow cytometry revealed the numbers of CD4+ and CD8+ T cells were increased in blood, lymphoid organs, and retina throughout the development of neovascular retinopathy. Interestingly, the depletion of CD8+ T cells but not CD4+ T cells reduced retinal neovascularization and vascular leakage. Using reporter mice expressing gfp (green fluorescence protein) in CD8+ T cells, these cells were localized near neovascular tufts in the retina, confirming that CD8+ T cells contribute to the disease. Furthermore, the adoptive transfer of CD8+ T cells deficient in TNF (tumor necrosis factor), IFNγ (interferon gamma), Prf (perforin), or GzmA/B (granzymes A/B) into immunocompetent Rag1-/- mice revealed that CD8+ T cells mediate retinal vascular disease via these factors, with TNF influencing all aspects of vascular pathology. The pathway by which CD8+ T cells migrate into the retina was identified as CXCR3 (C-X-C motif chemokine receptor 3) with the CXCR3 blockade reducing the number of CD8+ T cells within the retina and retinal vascular disease. CONCLUSIONS: We discovered that CXCR3 is central to the migration of CD8+ T cells into the retina as the CXCR3 blockade reduced the number of CD8+ T cells within the retina and vasculopathy. This research identified an unappreciated role for CD8+ T cells in retinal inflammation and vascular disease. Reducing CD8+ T cells via their inflammatory and recruitment pathways is a potential treatment for neovascular retinopathies.
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Enfermedades de la Retina , Enfermedades Vasculares , Animales , Ratones , Linfocitos T CD8-positivos/metabolismo , Neovascularización Patológica , Retina/metabolismo , Enfermedades de la Retina/metabolismo , Interferón gamma/metabolismo , Enfermedades Vasculares/patología , Ratones Endogámicos C57BLRESUMEN
Phagocytes migrate into tissues to combat infection and maintain tissue homeostasis. As dysregulated phagocyte migration and function can lead to inflammation or susceptibility to infection, identifying molecules that control these processes is critical. Here, we show that the tetraspanin CD82 restrains the migration of neutrophils and macrophages into tissues. Cd82 -/- phagocytes exhibited excessive migration during in vivo models of peritoneal inflammation, superfusion of CXCL1, retinopathy of prematurity, and infection with the protozoan parasite L. mexicana. However, with the latter, while Cd82 -/- macrophages infiltrated infection sites at higher proportions, cutaneous L. mexicana lesions were larger and persisted, indicating a failure to control infection. Analyses of in vitro bone-marrow-derived macrophages showed CD82 deficiency altered cellular morphology, and impaired gene expression and metabolism in response to anti-inflammatory activation. Altogether, this work reveals an important role for CD82 in restraining phagocyte infiltration and mediating their differentiation in response to stimulatory cues.
RESUMEN
Reactive oxygen species (ROS) normally play an important physiological role in health regulating cellular processes and signal transduction. The amount of ROS is usually kept in fine balance with the generation of ROS largely being offset by the body's antioxidants. A tipping of this balance has increasingly been recognised as a contributor to human disease. The retina, as a result of its cellular anatomy and physical location, is a potent generator of ROS that has been linked to several major retinal diseases. This review will provide a summary of the role of oxidative stress in the pathogenesis of diabetic retinopathy, age-related macular degeneration, myopia, retinal vein occlusion, retinitis pigmentosa and retinopathy of prematurity. Therapies aimed at controlling oxidative stress in these diseases are also examined.
Asunto(s)
Antioxidantes/metabolismo , Estrés Oxidativo , Especies Reactivas de Oxígeno/metabolismo , Retina/patología , Enfermedades de la Retina/metabolismo , Animales , Humanos , Retina/metabolismo , Enfermedades de la Retina/patología , Transducción de SeñalRESUMEN
Bronchopulmonary dysplasia (BPD) and retinopathy of prematurity (ROP) are two debilitating disorders that develop in preterm infants exposed to supplemental oxygen to prevent respiratory failure. Both can lead to lifelong disabilities, such as chronic obstructive pulmonary disease and vision loss. Due to the lack of a standard experimental model of coincident disease, the underlying associations between BPD and ROP are not well characterized. To address this gap, we used the robust mouse model of oxygen-induced retinopathy exposing C57BL/6 mice to 75% oxygen from postnatal day 7 to 12. The cardinal features of ROP were replicated by this strategy, and the lungs of the same mice were simultaneously examined for evidence of BPD-like lung injury, investigating both the short- and long-term effects of early-life supplemental oxygen exposure. At postnatal days 12 and 18, mild lung disease was evident by histopathologic analysis together with the expected vasculopathy in the inner retina. At later time points, the lung lesion had progressed to severe airspace enlargement and alveolar simplification, with concurrent thinning in the outer layer of the retina. In addition, critical angiogenic oxidative stress and inflammatory factors reported to be dysregulated in ROP were similarly impaired in the lungs. These data shed new light on the interconnectedness of these two neonatal disorders, holding potential for the discovery of novel targets to treat BPD and ROP.
Asunto(s)
Displasia Broncopulmonar/etiología , Modelos Animales de Enfermedad , Terapia por Inhalación de Oxígeno/efectos adversos , Oxígeno/toxicidad , Retinopatía de la Prematuridad/etiología , Animales , Animales Recién Nacidos , Displasia Broncopulmonar/patología , Inflamación/etiología , Inflamación/patología , Ratones , Ratones Endogámicos C57BL , Estrés Oxidativo/fisiología , Retinopatía de la Prematuridad/patologíaRESUMEN
Hypertension is a risk factor for the vascular permeability and neovascularization that threatens vision in diabetic retinopathy. Excess reactive oxygen species derived from the Nox (NADPH oxidase) isoforms, Nox1 and Nox4, contributes to vasculopathy in diabetic retinopathy; however, if Nox1/4 inhibition is beneficial in hypertensive diabetic retinopathy is unknown. Here, we determined that diabetic spontaneously hypertensive rats had exacerbated retinal vascular permeability and expression of angiogenic and inflammatory factors, compared with normotensive diabetic Wistar Kyoto rats. GKT136901, a specific dual inhibitor of Nox1 and Nox4, prevented these events in diabetic Wistar Kyoto rats and spontaneously hypertensive rats. Retinal neovascularization does not develop in diabetic rodents, and therefore, the oxygen-induced retinopathy model is used to evaluate this pathology. We previously demonstrated that Nox1/4 inhibition reduced retinal neovascularization in oxygen-induced retinopathy. However, although Nox5 is expressed in human retina, its contribution to retinopathy has not been studied in vivo, largely due to its absence from the rodent genome. We generated transgenic mice with inducible human Nox5 expressed in endothelial cells (vascular endothelial-cadherin+Nox5+ mice). In vascular endothelial-cadherin+Nox5+ mice with oxygen-induced retinopathy, retinal vascular permeability and neovascularization, as well as the expression of angiogenic and inflammatory factors, were increased compared with wild-type littermates. In bovine retinal endothelial cells, which express Nox1, Nox4, and Nox5, Nox1/4 inhibition, as well as Nox5 silencing RNA, reduced the high glucose-induced upregulation of oxidative stress, angiogenic, and inflammatory factors. Collectively, these data indicate the potential of Nox1, Nox4, and Nox5 inhibition to reduce vision-threatening damage to the retinal vasculature.
Asunto(s)
Permeabilidad Capilar/genética , Diabetes Mellitus Experimental/metabolismo , Retinopatía Diabética/metabolismo , NADPH Oxidasa 1/metabolismo , NADPH Oxidasa 4/metabolismo , NADPH Oxidasa 5/metabolismo , Neovascularización Retiniana/metabolismo , Animales , Glucemia , Presión Sanguínea/fisiología , Peso Corporal/fisiología , Bovinos , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/patología , Retinopatía Diabética/genética , Retinopatía Diabética/patología , Células Endoteliales/metabolismo , Ratones , Ratones Transgénicos , NADPH Oxidasa 1/genética , NADPH Oxidasa 4/genética , NADPH Oxidasa 5/genética , Estrés Oxidativo/fisiología , Ratas , Retina/metabolismo , Retina/patología , Neovascularización Retiniana/genética , Neovascularización Retiniana/patologíaRESUMEN
Microglial cells are important contributors to the neuroinflammation and blood vessel damage that occurs in ischemic retinopathies. We hypothesized that key effectors of the renin-angiotensin aldosterone system, angiotensin II (Ang II) and aldosterone, increase the density of microglia in the retina and stimulate their production of reactive oxygen species (ROS) as well as pro-angiogenic and pro-inflammatory factors. Two animal models were studied that featured up-regulation of Ang II or aldosterone and included transgenic Ren-2 rats which overexpress renin and Ang II in tissues including the retina, and Sprague Dawley rats with ischemic retinopathy and infused with aldosterone. Complementary studies were performed in primary cultures of retinal microglia from neonatal Sprague Dawley rats exposed to hypoxia (0.5% O2) and inhibitors of the angiotensin type 1 receptor (valsartan), the mineralocorticoid receptor (spironolactone) or aldosterone synthase (FAD286). In both in vivo models, the density of ionized calcium-binding adaptor protein-1 labelled microglia/macrophages was increased in retina compared to genetic or vehicle controls. In primary cultures of retinal microglia, hypoxia increased ROS (superoxide) levels as well as the expression of the NADPH oxidase (NOX) isoforms, NOX1, NOX2 and NOX4. The elevated levels of ROS as well as NOX2 and NOX4 were reduced by all of the treatments, and valsartan and FAD286 also reduced NOX1 mRNA levels. A protein cytokine array of retinal microglia revealed that valsartan, spironolactone and FAD286 reduced the hypoxia-induced increase in the potent pro-angiogenic and pro-inflammatory agent, vascular endothelial growth factor as well as the inflammatory factors, CCL5 and interferon γ. Valsartan also reduced the hypoxia-induced increase in IL-6 and TIMP-1 as well as the chemoattractants, CXCL2, CXCL3, CXCL5 and CXCL10. Spironolactone and FAD286 reduced the levels of CXCL2 and CXCL10, respectively. In conclusion, our findings that both Ang II and aldosterone influence the activation of retinal microglia implicates the renin-angiotensin aldosterone system in the pathogenesis of ischemic retinopathies.
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Aldosterona/farmacología , Angiotensina II/farmacología , Microglía/efectos de los fármacos , Neuronas Retinianas/efectos de los fármacos , Vasoconstrictores/farmacología , Animales , Animales Recién Nacidos , Células Cultivadas , Citocromo P-450 CYP11B2/metabolismo , Femenino , Inmunohistoquímica , Microglía/metabolismo , Oxígeno/toxicidad , Ratas , Ratas Sprague-Dawley , Ratas Transgénicas , Especies Reactivas de Oxígeno/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptor de Angiotensina Tipo 1/metabolismo , Receptores de Mineralocorticoides/metabolismo , Neovascularización Retiniana/etiología , Neovascularización Retiniana/metabolismo , Neovascularización Retiniana/patología , Neuronas Retinianas/metabolismo , Retinopatía de la Prematuridad/etiología , Retinopatía de la Prematuridad/metabolismo , Retinopatía de la Prematuridad/patologíaRESUMEN
Angiotensin II and aldosterone are the main effectors of the renin-angiotensin aldosterone system (RAAS) and have a central role in hypertension as well as cardiovascular and renal disease. The localization of RAAS components within the retina has led to studies investigating the roles of angiotensin II, aldosterone and the counter regulatory arm of the pathway in vision-threatening retinopathies. This review will provide a brief overview of RAAS components as well as the vascular pathology that develops in the retinal diseases, retinopathy of prematurity, diabetic retinopathy and neovascular age-related macular degeneration. The review will discuss pre-clinical and clinical evidence that modulation of the RAAS alters the development of vasculopathy and inflammation in the aforementioned retinopathies, as well as the emerging role of aldosterone and the mineralocorticoid receptor in central serous chorioretinopathy.
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Aldosterona/fisiología , Angiotensina II/fisiología , Retinopatía Diabética/fisiopatología , Vasos Retinianos/fisiología , Retinitis/fisiopatología , Retinopatía de la Prematuridad/fisiopatología , Degeneración Macular Húmeda/fisiopatología , Inhibidores de la Enzima Convertidora de Angiotensina , Humanos , Receptor de Angiotensina Tipo 1 , Sistema Renina-Angiotensina/fisiologíaRESUMEN
Hypertension is an independent risk factor for diabetic retinopathy, yet anti-hypertensive medications such as blockade of angiotensin II do not completely protect against vision-threatening vascular disease. We hypothesized that the potent vasoactive factor, endothelin (ET), is up-regulated in diabetic retinopathy and antagonism of the ET type A receptor (ETRA) or ET type B receptor (ETRB) ameliorates retinal vascular leakage independently of any blood pressure lowering effects. Spontaneously hypertensive rats (SHR) and their normotensive and genetic controls, Wistar Kyoto rats, were randomized to become diabetic or non-diabetic and studied for 8 weeks. Rats were further randomized to receive by intravitreal injection the ETRA antagonist, BQ123, the ETRB antagonist, BQ788, or vehicle, 5 days after the induction of streptozotocin diabetes and 4 weeks later. The treatments had no effect on systolic blood pressure which remained elevated in SHR. ET-1, ET-2, ETRA and ETRB were expressed in retina and retinal pigment epithelium (RPE)/choroid and increased by hypertension or diabetes. BQ123 reduced ET-1 and ET-2 expression in retina and RPE/choroid, while BQ788 had a similar effect but did not influence the mRNA levels of ET-1 in retina. Retinal vascular leakage and Müller cell stress as well as vascular endothelial growth factor (VEGF) expression in retina and RPE/choroid, were increased by hypertension or diabetes and there was an additive effect of these conditions. Treatment with BQ123 or BQ788 effectively reduced these events as well as the elevated levels of inflammatory factors in the retina. Our findings indicate that local ET systems exist in the retina and RPE/choroid that are up-regulated by hypertension and diabetes. The ability of locally delivered ET receptor antagonists to supress these overactive ET systems and reduce retinal vascular leakage and VEGF in the presence of hypertension indicate the potential of these approaches for the treatment of diabetic retinopathy.
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Antihipertensivos/uso terapéutico , Retinopatía Diabética/prevención & control , Antagonistas de los Receptores de la Endotelina A/uso terapéutico , Antagonistas de los Receptores de la Endotelina B/uso terapéutico , Hipertensión Ocular/prevención & control , Animales , Presión Sanguínea/efectos de los fármacos , Barrera Hematorretinal/efectos de los fármacos , Coroides/metabolismo , Diabetes Mellitus Experimental/prevención & control , Retinopatía Diabética/metabolismo , Antagonistas de los Receptores de la Endotelina A/metabolismo , Antagonistas de los Receptores de la Endotelina B/metabolismo , Endotelina-1/genética , Endotelina-1/metabolismo , Endotelina-2/genética , Endotelina-2/metabolismo , Ensayo de Inmunoadsorción Enzimática , Técnica del Anticuerpo Fluorescente Indirecta , Inyecciones Intravítreas , Hipertensión Ocular/metabolismo , Oligopéptidos/uso terapéutico , Péptidos Cíclicos/uso terapéutico , Piperidinas/uso terapéutico , Ratas , Ratas Endogámicas SHR , Ratas Endogámicas WKY , Reacción en Cadena en Tiempo Real de la Polimerasa , Retina/metabolismo , Epitelio Pigmentado de la Retina/metabolismo , EstreptozocinaRESUMEN
Purpose: Oxidative stress is a causal factor in the development of diabetic retinopathy; however, clinically relevant strategies to treat the disease by augmenting antioxidant defense mechanisms have not been fully explored. We hypothesized that boosting nuclear factor erythroid-2-related factor 2 (Nrf2) antioxidant capacity with the novel Nrf2 activator dh404, would protect the retina in diabetes including vision-threatening breakdown of the blood-retinal barrier (BRB) and associated damage to macroglial Müller cells. Methods: Sprague-Dawley rats were randomized to become diabetic or nondiabetic and administered dh404 by gavage for 10 weeks. Complementary in vitro studies were performed in cultured Müller cells exposed to hyperglycemia. Results: In diabetes, dh404 prevented vascular leakage into the retina and vitreous cavity as well as upregulation of the vascular permeability and angiogenic factors, VEGF, and angiopoietin-2, and inflammatory mediators, including TNF-α and IL-6. Müller cells, which maintain BRB integrity and become gliotic in diabetes with increased immunolabeling for glial fibrillary acidic protein, were protected by dh404. In diabetes, dh404 bolstered the antioxidant capacity of the retina with an increase in hemeoxygenase-1, nicotinamide adenine dinucleotide/nicotinamide adenine dinucleotide phosphate (NADH/NADPH) quinine oxidoreductase-1, and Nrf2. Further, dh404 attenuated the diabetes-induced increase in oxidative stress as measured by dihydroethidium and 8-oxo-2'-deoxyguanosine (8-OHdG) immunolabeling as well as NADPH oxidase isoform expression. Studies in Müller cells supported these findings with dh404 attenuating the hyperglycemia-induced increase in vascular permeability, angiogenic and inflammatory mediators, and oxidative stress. Conclusions: Our data demonstrate the ability of dh404 to protect the retina against diabetes-induced damage and potentially prevent vision loss.
Asunto(s)
Barrera Hematorretinal/efectos de los fármacos , Permeabilidad Capilar/efectos de los fármacos , Diabetes Mellitus Experimental/prevención & control , Retinopatía Diabética/prevención & control , Células Ependimogliales/efectos de los fármacos , Factor 2 Relacionado con NF-E2/metabolismo , Ácido Oleanólico/análogos & derivados , Angiopoyetina 2/metabolismo , Animales , Barrera Hematorretinal/fisiología , Western Blotting , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/patología , Retinopatía Diabética/metabolismo , Retinopatía Diabética/patología , Ensayo de Inmunoadsorción Enzimática , Células Ependimogliales/metabolismo , Gliosis , Interleucina-6/metabolismo , Ácido Oleanólico/farmacología , Estrés Oxidativo/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Reacción en Cadena en Tiempo Real de la Polimerasa , Factor de Necrosis Tumoral alfa/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
BACKGROUND: Over-production of reactive oxygen species (ROS) and resulting oxidative stress contribute to retinal damage in vascular diseases that include diabetic retinopathy, retinopathy of prematurity and major retinal vessel occlusions. NADPH oxidase (Nox) proteins are professional ROS-generating enzymes, and therapeutic targeting in these diseases has strong appeal. Pharmacological inhibition of Nox4 reduces the severity of experimental retinal vasculopathy. We investigated the potential application of this drug approach in humans. METHODS: Differential Nox enzyme expression was studied by real-time-quantitative polymerase chain reaction in primary human retinal endothelial cell isolates and a characterized human retinal endothelial cell line. Oxidative stress was triggered chemically in endothelial cells, by treatment with dimethyloxalylglycine (DMOG; 100 µM); Nox4 and vascular endothelial growth factor (VEGFA) transcript were measured; and production of ROS was detected by 2',7'-dichlorofluorescein. DMOG-stimulated endothelial cells were treated with two Nox1/Nox4 inhibitors, GKT136901 and GKT137831; cell growth was monitored by DNA quantification, in addition to VEGFA transcript and ROS production. RESULTS: Nox4 (isoform Nox4A) was the predominant Nox enzyme expressed by human retinal endothelial cells. Treatment with DMOG significantly increased endothelial cell expression of Nox4 over 72 h, accompanied by ROS production and increased VEGFA expression. Treatment with GKT136901 or GKT137831 significantly reduced DMOG-induced ROS production and VEGFA expression by endothelial cells, and the inhibitory effect of DMOG on cell growth. CONCLUSIONS: Our findings in experiments on activated human retinal endothelial cells provide translational corroboration of studies in experimental models of retinal vasculopathy and support the therapeutic application of Nox4 inhibition by GKT136901 and GKT137831 in patients with retinal vascular diseases.
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Retinopatía Diabética/genética , Células Endoteliales/metabolismo , Regulación de la Expresión Génica , NADPH Oxidasa 1/genética , Estrés Oxidativo , Vasos Retinianos/patología , Proliferación Celular , Células Cultivadas , Retinopatía Diabética/metabolismo , Retinopatía Diabética/patología , Células Endoteliales/patología , Humanos , NADPH Oxidasa 1/biosíntesis , ARN/genética , Especies Reactivas de Oxígeno/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Vasos Retinianos/metabolismoRESUMEN
Diabetes is a negative risk factor for aortic aneurysm, but the underlying explanation for this phenomenon is unknown. We have previously demonstrated that cell division autoantigen 1 (CDA1), which enhances transforming growth factor-ß signaling, is upregulated in diabetes. We hypothesized that CDA1 plays a key role in conferring the protective effect of diabetes against aortic aneurysms. Male wild-type, CDA1 knockout (KO), apolipoprotein E (ApoE) KO, and CDA1/ApoE double-KO (dKO) mice were rendered diabetic. Whereas aneurysms were not observed in diabetic ApoE KO and wild-type mice, 40% of diabetic dKO mice developed aortic aneurysms. These aneurysms were associated with attenuated aortic transforming growth factor-ß signaling, reduced expression of various collagens, and increased aortic macrophage infiltration and matrix metalloproteinase 12 expression. In the well-characterized model of angiotensin II-induced aneurysm formation, concomitant diabetes reduced fatal aortic rupture and attenuated suprarenal aortic expansion, changes not seen in dKO mice. Furthermore, aortic CDA1 expression was downregulated â¼70% within biopsies from human abdominal aortic aneurysms. The identification that diabetes is associated with upregulation of vascular CDA1 and that CDA1 deletion in diabetic mice promotes aneurysm formation provides evidence that CDA1 plays a role in diabetes to reduce susceptibility to aneurysm formation.
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Aneurisma de la Aorta Abdominal/genética , Autoantígenos/genética , Diabetes Mellitus Experimental/metabolismo , Adulto , Anciano , Angiotensina II/farmacología , Animales , Aneurisma de la Aorta/inducido químicamente , Aneurisma de la Aorta/genética , Aneurisma de la Aorta/inmunología , Aneurisma de la Aorta/metabolismo , Aneurisma de la Aorta Abdominal/inducido químicamente , Aneurisma de la Aorta Abdominal/inmunología , Aneurisma de la Aorta Abdominal/metabolismo , Rotura de la Aorta , Autoantígenos/metabolismo , Colágeno/metabolismo , Femenino , Regulación de la Expresión Génica , Humanos , Macrófagos/inmunología , Masculino , Metaloproteinasa 12 de la Matriz/metabolismo , Ratones , Ratones Noqueados , Ratones Noqueados para ApoE , Persona de Mediana Edad , Índice de Severidad de la Enfermedad , Transducción de Señal , Proteína smad3/metabolismo , Factor de Crecimiento Transformador beta/inmunología , Factor de Crecimiento Transformador beta/metabolismo , Vasoconstrictores/farmacologíaRESUMEN
Although increasing evidence indicates that endothelin-2 (Edn2) has distinct roles in tissue pathology, including inflammation, glial cell dysfunction, and angiogenesis, its role in the retina and the factors that regulate its actions are not fully understood. We hypothesized that Edn2 damages the blood-retinal barrier (BRB) and that this is mediated by interactions with the renin-angiotensin-aldosterone system and reactive oxygen species derived from NADPH oxidase (Nox). C57BL/6J mice received an intravitreal injection of Edn2 or control vehicle to examine the blood pressure-independent effects of Edn2. Mice administered Edn2 were randomized to receive by intraperitoneal injection treatments that inhibited the Edn type a receptor, Edn type b receptor, angiotensin type 1 receptor, mineralocorticoid receptor, or Nox isoforms 1 to 4. One month later, mice administered Edn2 exhibited breakdown of the BRB with increased vascular leakage, vascular endothelial growth factor expression, and infiltrating macrophages (Ly6C+CD45highCD11b+). Further, macroglial Müller cells, which influence the integrity of the BRB and prevent retinal edema, became gliotic and expressed increased levels of water (aquaporin-4) and ion (Kir4.1) channels. This Edn2-mediated retinopathy was reduced by all treatments. Complementary in vitro studies in cultured Müller cells supported these findings and demonstrated the importance of reactive oxygen species in mediating these events. In conclusion, Edn2 has detrimental effects on the BRB and Müller cells that involve interactions with the renin-angiotensin aldosterone system and Nox1/4.
Asunto(s)
Aldosterona/farmacología , Angiotensina II/farmacología , Barrera Hematorretinal/efectos de los fármacos , Endotelina-2/farmacología , Células Ependimogliales/efectos de los fármacos , NADPH Oxidasas/metabolismo , Retina/efectos de los fármacos , Acuaporina 4/metabolismo , Barrera Hematorretinal/metabolismo , Barrera Hematorretinal/patología , Movimiento Celular/efectos de los fármacos , Células Ependimogliales/metabolismo , Células Ependimogliales/patología , Humanos , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Macrófagos/patología , Especies Reactivas de Oxígeno/metabolismo , Retina/metabolismo , Retina/patologíaRESUMEN
For many years, oxidative stress arising from the ubiquitous production of reactive oxygen species (ROS) has been implicated in the pathogenesis of various eye diseases. While emerging research has provided some evidence of the important physiological role of ROS in normal cell function, disease may arise where the concentration of ROS exceeds and overwhelms the body's natural defence against them. Additionally, ROS may induce genomic aberrations which affect cellular homoeostasis and may result in disease. This literature review examines the current evidence for the role of oxidative stress in important ocular diseases with a view to identifying potential therapeutic targets for future study. The need is particularly pressing in developing treatments for conditions which remain notoriously difficult to treat, including glaucoma, diabetic retinopathy and age-related macular degeneration.
Asunto(s)
Oftalmopatías/metabolismo , Ojo/metabolismo , Estrés Oxidativo , Especies Reactivas de Oxígeno/metabolismo , Animales , Antioxidantes/uso terapéutico , Ojo/efectos de los fármacos , Ojo/patología , Oftalmopatías/tratamiento farmacológico , Oftalmopatías/patología , Humanos , Estrés Oxidativo/efectos de los fármacos , Transducción de SeñalRESUMEN
Cell membrane thyroid hormone (TH) transport can be facilitated by the monocarboxylate transporter 8 (MCT8), encoded by the solute carrier family 16 member 2 (SLC16A2) gene. Human mutations of the gene, SLC16A2, result in the X-linked-inherited psychomotor retardation and hypomyelination disorder, Allan-Herndon-Dudley syndrome (AHDS). We posited that abrogating MCT8-dependent TH transport limits oligodendrogenesis and myelination. We show that human oligodendrocytes (OL), derived from the NKX2.1-GFP human embryonic stem cell (hESC) reporter line, express MCT8. Moreover, treatment of these cultures with DITPA (an MCT8-independent TH analog), up-regulates OL differentiation transcription factors and myelin gene expression. DITPA promotes hESC-derived OL myelination of retinal ganglion axons in co-culture. Pharmacological and genetic blockade of MCT8 induces significant OL apoptosis, impairing myelination. DITPA treatment limits OL apoptosis mediated by SLC16A2 down-regulation primarily signaling through AKT phosphorylation, driving myelination. Our results highlight the potential role of MCT8 in TH transport for human OL development and may implicate DITPA as a promising treatment for developmentally-regulated myelination in AHDS.
Asunto(s)
Diferenciación Celular/genética , Discapacidad Intelectual Ligada al Cromosoma X/genética , Transportadores de Ácidos Monocarboxílicos/genética , Hipotonía Muscular/genética , Atrofia Muscular/genética , Neurogénesis/genética , Transporte Biológico/genética , Línea Celular , Diyodotironinas/administración & dosificación , Células Madre Embrionarias Humanas/efectos de los fármacos , Humanos , Discapacidad Intelectual Ligada al Cromosoma X/tratamiento farmacológico , Discapacidad Intelectual Ligada al Cromosoma X/patología , Hipotonía Muscular/tratamiento farmacológico , Hipotonía Muscular/patología , Atrofia Muscular/tratamiento farmacológico , Atrofia Muscular/patología , Mutación , Vaina de Mielina/metabolismo , Oligodendroglía/metabolismo , Oligodendroglía/patología , Propionatos/administración & dosificación , Simportadores , Hormonas Tiroideas/genética , Hormonas Tiroideas/metabolismoRESUMEN
Neovascular retinopathies are major causes of vision loss; yet treatments to prevent the condition are inadequate. The role of regulatory T cells in neovascular retinopathy is unknown. Here we show that in retinopathy regulatory T cells are transiently increased in lymphoid organs and the retina, but decline when neovascularization is established. The decline is prevented following regulatory T cells expansion with an IL-2/anti-IL-2 mAb complex or the adoptive transfer of regulatory T cells. Further, both approaches reduce vasculopathy (vaso-obliteration, neovascularization, vascular leakage) and alter the activation of Tmem119+ retinal microglia. Our in vitro studies complement these findings, showing that retinal microglia co-cultured with regulatory T cells exhibit a reduction in co-stimulatory molecules and pro-inflammatory mediators that is attenuated by CTLA-4 blockade. Collectively, we demonstrate that regulatory T cells are recruited to the retina and, when expanded in number, repair the vasculature. Manipulation of regulatory T cell numbers is a previously unrecognized, and promising avenue for therapies to prevent blinding neovascular retinopathies.The local immune responses in the eye are attenuated to preserve sight. Surprisingly, Deliyanti et al. show that regulatory T cells (Tregs) take an active role in protecting the eye from neovascularization in oxygen-induced retinopathy, and that interventions that augment the retinal Treg numbers reduce neovascular retinopathy in mice.