RESUMEN
Epigenetic deregulation is increasingly recognized as a contributing pathological factor in multiple myeloma (MM). In particular tri-methylation of H3 lysine 27 (H3K27me3), which is catalyzed by PHD finger protein 19 (PHF19), a subunit of the Polycomb Repressive Complex 2 (PRC2), has recently shown to be a crucial mediator of MM tumorigenicity. Overexpression of PHF19 in MM has been associated with worse clinical outcome. Yet, while there is mounting evidence that PHF19 overexpression plays a crucial role in MM carcinogenesis downstream mechanisms remain to be elucidated. In the current study we use a functional knock down (KD) of PHF19 to investigate the biological role of PHF19 and show that PHF19KD leads to decreased tumor growth in vitro and in vivo. Expression of major cancer players such as bcl2, myc and EGR1 were decreased upon PHF19KD further underscoring the role of PHF19 in MM biology. Additionally, our results highlighted the prognostic impact of PHF19 overexpression, which was significantly associated with worse survival. Overall, our study underscores the premise that targeting the PHF19-PRC2 complex would open up avenues for novel MM therapies.
Asunto(s)
Mieloma Múltiple , Proliferación Celular , Proteínas de Unión al ADN , Humanos , Mieloma Múltiple/tratamiento farmacológico , Mieloma Múltiple/genética , Complejo Represivo Polycomb 2 , Factores de Transcripción/genéticaRESUMEN
In the mouse, membrane cofactor protein (CD46), a key regulator of the alternative pathway of the complement system, is only expressed in the eye and on the inner acrosomal membrane of spermatozoa. We noted that although Cd46(-/-) mice have normal systemic alternative pathway activating ability, lack of CD46 leads to dysregulated complement activation in the eye, as evidenced by increased deposition of C5b-9 in the retinal pigment epithelium (RPE) and choroid. A knockout of CD46 induced the following cardinal features of human dry age-related macular degeneration (AMD) in 12-month-old male and female mice: accumulation of autofluorescent material in and hypertrophy of the RPE, dense deposits in and thickening of Bruch's membrane, loss of photoreceptors, cells in subretinal space, and a reduction of choroidal vessels. Collectively, our results demonstrate spontaneous age-related degenerative changes in the retina, RPE, and choroid of Cd46(-/-) mice that are consistent with human dry AMD. These findings provide the exciting possibility of using Cd46(-/-) mice as a convenient and reliable animal model for dry AMD. Having such a relatively straight-forward model for dry AMD should provide valuable insights into pathogenesis and a test model system for novel drug targets. More important, tissue-specific expression of CD46 gives the Cd46(-/-) mouse model of dry AMD a unique advantage over other mouse models using knockout strains.
Asunto(s)
Modelos Animales de Enfermedad , Atrofia Geográfica/genética , Degeneración Macular/genética , Proteína Cofactora de Membrana/deficiencia , Animales , Western Blotting , Femenino , Atrofia Geográfica/patología , Degeneración Macular/patología , Masculino , Proteína Cofactora de Membrana/genética , Ratones , Ratones NoqueadosRESUMEN
BACKGROUND: To characterize the effect of aspirin (ASA) in mouse models of choroidal neovascularization (CNV) and retinal degeneration. METHODS: In vivo: Male C57BL/6 mice were given ASA in food or regular rodent diet. CNV was induced by argon laser photocoagulation. Subretinal injections of polyethylene glycol 400 (PEG-400) were administered to induce retinal degeneration. CNV size, laser spot area and mean intensity of VEGF in the laser injured zones were measured. In the PEG injected eyes the thickness of retinal pigment epithelium (RPE) and choroid was measured. In vitro: Human ARPE-19 cells were treated with 0.5 or 2.0 mM/L of ASA for 72 h. ELISA was used to measure the concentration of VEGF and CCL-2 in the supernatants. Additionally, damaged RPE monolayer was treated with ASA (0.5 or 2.0 mM/L) and vehicle separately. Size of damaged area was measured. ELISA was used to measure secretion of VEGF-A and CCL-2 by damaged cells after 24 h. RESULTS: No statistically significant effect of ASA on CNV size, laser spot size or VEGF expression was noted in CNV model. In the PEG model, ASA did not have any effect on RPE and choroid thickness; however, a significant increase in RPE atrophy was observed (P = 0.02 + 38%). In addition, ASA had a significant effect on the ability of the RPE cells to regenerate and become confluent after mechanical damage. CONCLUSIONS: ASA at doses consumed clinically for various medical causes may not worsen CNV in human subjects. However, ASA may increase RPE atrophy when consumed over long periods of time.
Asunto(s)
Antiinflamatorios no Esteroideos/farmacología , Aspirina/farmacología , Neovascularización Coroidal/tratamiento farmacológico , Modelos Animales de Enfermedad , Degeneración Retiniana/tratamiento farmacológico , Epitelio Pigmentado de la Retina/efectos de los fármacos , Animales , Línea Celular , Quimiocina CCL2/metabolismo , Neovascularización Coroidal/metabolismo , Neovascularización Coroidal/patología , Dieta , Ensayo de Inmunoadsorción Enzimática , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Degeneración Retiniana/metabolismo , Degeneración Retiniana/patología , Epitelio Pigmentado de la Retina/metabolismo , Epitelio Pigmentado de la Retina/patología , Factor A de Crecimiento Endotelial Vascular/metabolismo , Cicatrización de Heridas/efectos de los fármacosRESUMEN
Studies performed over the past decade in humans and experimental animals have been a major source of information and improved our understanding of how dysregulation of the complement system contributes to age-related macular degeneration (AMD) pathology. Drusen, the hall-mark of dry-type AMD are reported to be the by-product of complement mediated inflammatory processes. In wet AMD, unregulated complement activation results in increased production of angiogenic growth factors leading to choroidal neovascularization both in humans and in animal models. In this review article we have linked the complement system with modifiable and non-modifiable AMD risk factors as well as with prediction models of AMD. Understanding the association between the complement system, risk factors and prediction models will help improve our understanding of AMD pathology and management of this disease.
Asunto(s)
Proteínas del Sistema Complemento/inmunología , Degeneración Macular/inmunología , Modelos Biológicos , Animales , Modelos Animales de Enfermedad , Humanos , Factores de RiesgoRESUMEN
Age-related macular degeneration (AMD) is a leading cause of irreversible blindness. This study was done to characterize dry AMD-like changes in mouse retinal pigment epithelium (RPE) and retina after polyethylene glycol (PEG) treatment. We injected male C57BL/6 mice subretinally with PBS, 0.025, 0.25, 0.5 and 1.0 mg of PEG-400 and the animals were sacrificed on day 5. Eyes were harvested and processed for histological analysis. In all other experiments 0.5 mg PEG was injected and animals were sacrificed on days 1, 3, 5 or 14. Paraffin, 5 µm and plastic, 1 µm and 80 nm sections were used for further analysis. Subretinal injection of 0.5 mg PEG induced a 32% reduction of outer nuclear layer (ONL) thickness, 61% decrease of photoreceptor outer and inner segment length, 49% decrease of nuclear density in the ONL and 31% increase of RPE cell density by day 5 after injection. The maximum level of TUNEL positive nuclei in the ONL (6.8 + 1.99%) was detected at day 5 after PEG injection and co-localized with Casp3act. Histological signs of apoptosis were observed in the ONL by light or electron microscopy. Degeneration of RPE cells was found in PEG injected eyes. Gene expression data identified several genes reported to be involved in human AMD. C3, Cfi, Serping1, Mmp9, Htra1 and Lpl were up-regulated in PEG injected eyes compared to PBS controls. PEG leads to morphological and gene expression changes in RPE and retina consistent with dry AMD. This model will be useful to investigate dry AMD pathogenesis and treatment.
Asunto(s)
Modelos Animales de Enfermedad , Atrofia Geográfica/patología , Células Fotorreceptoras de Vertebrados/patología , Polietilenglicoles/toxicidad , Epitelio Pigmentado de la Retina/patología , Aminopeptidasas/genética , Animales , Apoptosis , Autofagia , Proteína Inhibidora del Complemento C1/genética , Complemento C3/genética , Relación Dosis-Respuesta a Droga , Regulación de la Expresión Génica/fisiología , Atrofia Geográfica/inducido químicamente , Atrofia Geográfica/genética , Serina Peptidasa A1 que Requiere Temperaturas Altas , Etiquetado Corte-Fin in Situ , Inyecciones Intraoculares , Masculino , Metaloproteinasa 9 de la Matriz/genética , Ratones , Ratones Endogámicos C57BL , Serina Endopeptidasas/genética , Factores de TiempoRESUMEN
Age-related macular degeneration (AMD) is a leading cause of central blindness in the elderly population. The wet type of AMD is characterized by extensive growth of new vessels. One of the effective strategies to treat wet AMD is to limit the choroidal neovascularization (CNV). We studied the effects of adiponectin peptide I (APNpI) on new vessel growth in laser-induced rat model of wet AMD and on rat choroidal endothelial cell (CEC) culture. CNV size and vessel density were investigated by microscopy. Immunohistochemical staining (IHC) for von Willebrand Factor (vWF), APN, APN receptors 1 (AdipoR1), 2 (AdipoR2), VEGF, VEGF receptor 2 (VEGF-R2), proliferating cell nuclear antigen (PCNA) was performed in CNV area. The mRNA expression of VEGF and VEGF-R2 in RPE-choroid was investigated by RT-PCR and real-time PCR. APNpI inhibited area of CNV by 4 fold, number of vWF positive vessels by 99% and area of subretinal tissue by 40%. The expression of VEGF and VEGF-R2 at mRNA and protein levels decreased after APNpI treatment in vivo. Proliferative index (PCNA) was 5 folds less in laser spots of APNpI treated rats compared to controls. In conclusion, APNpI inhibited formation of new vessels in rat model of CNV by decreasing VEGF, VEGF-R2 expression and cell proliferation. Thus, APNpI may have potential therapeutic use for AMD treatment since it significantly inhibited CNV.
Asunto(s)
Adiponectina/farmacología , Coroides/irrigación sanguínea , Neovascularización Coroidal/prevención & control , Fragmentos de Péptidos/farmacología , Degeneración Macular Húmeda/tratamiento farmacológico , Adiponectina/uso terapéutico , Animales , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Coroides/efectos de los fármacos , Coroides/patología , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Células Endoteliales/fisiología , Expresión Génica/efectos de los fármacos , Masculino , Fragmentos de Péptidos/uso terapéutico , Antígeno Nuclear de Célula en Proliferación/metabolismo , Ratas Endogámicas BN , Receptores de Adiponectina/metabolismo , Factor A de Crecimiento Endotelial Vascular/genética , Factor A de Crecimiento Endotelial Vascular/metabolismo , Receptor 2 de Factores de Crecimiento Endotelial Vascular/genética , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismoRESUMEN
PURPOSE: The purpose of this article is to evaluate the effect of nicotine on anti-vascular endothelial growth factor therapy in the treatment of neovascular age-related macular degeneration. METHODS: One group of mice received nicotine in drinking water and the other group received water only. Choroidal neovascularization (CNV) was induced with a laser. Nicotinic acetylcholine receptor-α7 (nAChRα7) expression was evaluated by immunohistochemistry. Bevacizumab or adiponectin peptide II (APNpII) was injected intravitreally on Day 7 postlaser, and the effects were evaluated on Days 14 and 21. α-Bungarotoxin was injected intraperitoneally on Days 2 to 5, and its effect was evaluated on Day 14. RESULTS: Expression of nAChRα7 was 2 to 7 times higher between Days 3 and 7 postlaser compared with naive mice. In water-fed mice, APNpII, bevacizumab, and α-bungarotoxin significantly reduced CNV size. In nicotine-fed mice, treatment with APNpII or bevacizumab did not significantly reduce CNV size, whereas α-bungarotoxin did have an effect. Comparing water- and nicotine-fed mice, CNV size was 61% to 86% smaller in water-fed mice except for the α-bungarotoxin group, where there was no difference. Platelet-derived growth factor and vascular endothelial growth factor expression was 1.5- to 2.5-fold higher at Day 14 in nicotine-treated mice. CONCLUSION: Nicotine significantly blocks the effect of anti-vascular endothelial growth factor therapy in the treatment of laser-induced neovascular age-related macular degeneration. nAChRα7 is significantly upregulated during the formation of CNV, and treatment with an nAChRα7 antagonist decreases CNV size irrespective of nicotine administration.
Asunto(s)
Inhibidores de la Angiogénesis/uso terapéutico , Anticuerpos Monoclonales Humanizados/uso terapéutico , Neovascularización Coroidal/tratamiento farmacológico , Nicotina/farmacología , Factores de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Adiponectina/uso terapéutico , Análisis de Varianza , Animales , Bevacizumab , Bungarotoxinas/farmacología , Coroides/metabolismo , Neovascularización Coroidal/metabolismo , Neovascularización Coroidal/patología , Modelos Animales de Enfermedad , Inmunohistoquímica , Inyecciones Intravítreas , Masculino , Ratones , Ratones Endogámicos C57BL , Receptores Nicotínicos/metabolismo , Epitelio Pigmentado de la Retina/metabolismo , Factores de Crecimiento Endotelial Vascular/metabolismo , Receptor Nicotínico de Acetilcolina alfa 7RESUMEN
The present study investigated the interactions among the complement membrane attack complex (MAC), CCL2, and VEGF that occur in vivo during the development of choroidal neovascularization (CNV). We first investigated the sequential expression of MAC, CCL2, and VEGF during laser-induced CNV in C57BL/6 mice. Increased MAC deposition was detected at 1 h, CCL2 increased at 3 h, and VEGF was up-regulated at day 3 post-laser treatment. These results suggested that during laser-induced CNV, MAC, CCL2 and VEGF are formed and/or expressed in the following order: MAC â CCL2 â VEGF. To determine the cross-talk between MAC, CCL2, and VEGF during laser-induced CNV, neutralizing antibodies were injected both systemically and locally to block the bioactivity of each molecule. Blocking MAC formation inhibited CCL2 and VEGF expression and also limited CNV formation, whereas neutralization of CCL2 bioactivity did not affect MAC deposition; however, it reduced VEGF expression and CNV formation. When bioactivity of VEGF was blocked, CNV formation was significantly inhibited, but MAC deposition was not affected. Together, our results demonstrate that MAC is an upstream mediator and effect of MAC on the development of laser-induced CNV can be attributed to its direct effect on VEGF as well as its effect on VEGF that is mediated by CCL2. Understanding the interplay between immune mediators is critical to gain insight into the pathogenesis of CNV.
Asunto(s)
Quimiocina CCL2/biosíntesis , Neovascularización Coroidal/metabolismo , Complejo de Ataque a Membrana del Sistema Complemento/metabolismo , Regulación de la Expresión Génica , Rayos Láser/efectos adversos , Factor A de Crecimiento Endotelial Vascular/biosíntesis , Animales , Anticuerpos Neutralizantes/farmacología , Neovascularización Coroidal/etiología , Neovascularización Coroidal/patología , Modelos Animales de Enfermedad , Ratones , Factores de TiempoRESUMEN
In this study, we describe a new method for inducing choroidal neovascularization (CNV) in C57BL/6 mice, an animal model of wet age-related macular degeneration (AMD). AMD is a disease that causes central blindness in humans. We injected PEG-8 subretinally in different doses (0.125-2 mg) to induce CNV. After PEG-8 injection, we examined CNV at several time points (days 3-42). We also used Western blotting, immunohistochemistry, and ELISA to examine the complement component C3 split products, C9, VEGF, TGF-ß2, and basic FGF. As early as day 1 after treatment, we found that a single subretinal injection of 1 mg of PEG-8 increased the C3 split products and the C9, TGF-ß2, and basic FGF levels in the retinal pigment epithelium-choroid tissue. By day 3 after PEG-8 injection, the intraocular activation of the complement system caused induction and progression of CNV, including new vessels penetrating the Bruch's membrane. At day 5 after PEG-8 injection, we observed a fully developed CNV and retinal degeneration. Thus, in this study, we present a new, inexpensive, and accelerated mouse model of CNV that may be useful to study AMD.
Asunto(s)
Neovascularización Coroidal/inducido químicamente , Modelos Animales de Enfermedad , Portadores de Fármacos/efectos adversos , Degeneración Macular , Polietilenglicoles/efectos adversos , Animales , Neovascularización Coroidal/metabolismo , Neovascularización Coroidal/patología , Complemento C3/metabolismo , Complemento C9/metabolismo , Portadores de Fármacos/farmacología , Ratones , Polietilenglicoles/farmacología , Factores de Tiempo , Factor de Crecimiento Transformador beta2/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
The objective of this study was to explore the relationship between local (ie, ocular) complement factor H (CFH) and choroidal neovascularization (CNV) associated with wet age-related macular degeneration (AMD), a leading cause of irreversible blindness, in laser-treated C57BL/6 mice. Immunohistochemical and RT-PCR analysis of retinal pigmented epithelium (RPE)-choroid sclera revealed that the expression of CFH was down-regulated on day 1 with a dramatic increase on days 5 and 7 postlaser injury. Flat mount and Western blot analysis further revealed that membrane attack complex (MAC) expression was up-regulated on days 1 and 3 postlaser injury; however, MAC was down-regulated on days 5 and 7 postinjury but was still higher than in non-injured mice. Similar patterns for CFH and MAC were observed for RPE cells when serial paraffin sections of the laser spots were analyzed. Subretinal injection of siRNA directed against CFH resulted in a threefold suppression of CFH in the RPE and choroid without affecting either CFH levels in the liver or the functional activity of the alternative pathway in the peripheral blood. Ocular knock-down of CFH resulted in increased MAC deposition, which leads to the early onset as well as exacerbation of laser-induced CNV. In conclusion, our findings provide evidence that CFH present on RPE and choroid regulates local MAC formation that is critical for the development of laser-induced CNV.
Asunto(s)
Neovascularización Coroidal/etiología , Neovascularización Coroidal/metabolismo , Factor H de Complemento/fisiología , Modelos Animales de Enfermedad , Rayos Láser/efectos adversos , Epitelio Pigmentado Ocular/metabolismo , Animales , Western Blotting , Neovascularización Coroidal/patología , Factor H de Complemento/antagonistas & inhibidores , Técnicas para Inmunoenzimas , Degeneración Macular/etiología , Degeneración Macular/metabolismo , Degeneración Macular/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Epitelio Pigmentado Ocular/patología , ARN Mensajero/genética , ARN Interferente Pequeño/farmacología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Visión Ocular/fisiologíaRESUMEN
This study was designed to explore the effect of recombinant, membrane-targeted CD59 (rCD59-APT542) on the growth and size of fully developed neovascular complex using the murine model of laser-induced choroidal neovascularization (CNV). CNV was induced by laser photocoagulation in C57BL/6 mice using an argon laser, and the animals received rCD59-APT542 via intravitreal (ivt) route. Western blot analysis, immunohistochemistry, and total complement hemolytic assay demonstrated that exogenously administered rCD59-APT542 was incorporated as well as retained in RPE and choroid and was functionally active in vivo. Single ivt injection during the growth of the CNV (i.e. at day 3 post-laser) resulted in â¼79% inhibition of the further growth of neovascular complex. The size of the CNV complex was significantly (p < 0.05) reduced by the administration of rCD59-APT542 after the CNV complex has fully developed (i.e. at day 7 post-laser). Treatment with rCD59-APT542 blocked the formation of membrane attack complex (MAC), increased apoptosis and decreased cell proliferation in the neovascular complex. On the basis of results presented here we conclude that recombinant membrane targeted CD59 inhibited the growth of the CNV complex and reduced the size of fully developed CNV in the laser-induced mouse model. We propose that a combination of two mechanisms: increased apoptosis and decreased cell proliferation, both resulting from local inhibition of MAC, may be responsible for inhibition of CNV by rCD59-APT542.
Asunto(s)
Antígenos CD59/metabolismo , Neovascularización Coroidal/metabolismo , Neovascularización Patológica/metabolismo , Proteínas Recombinantes/química , Animales , Apoptosis , Membrana Celular/metabolismo , Proliferación Celular , Proteínas del Sistema Complemento/química , Inmunohistoquímica , Inflamación , Degeneración Macular/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BLRESUMEN
We have investigated the effect of adiponectin (APN) peptide II on new vessel growth in mouse model of choroidal neovascularization (CNV) or wet type age-related macular degeneration (AMD). Mice were injected intraperitoneally with APN peptide II, control peptide, or PBS on day 1-7 or day 5-14. APN, AdipoR1, PCNA, and VEGF localization was investigated using confocal microscopy, immunohistochemistry, and RT-PCR. APN peptide II decreased the relative area of FITC-dextran perfused vessels by 4-fold, PCNA expression by 3-fold, and the number of PCNA stained HUVEC and MAVEC cells by 38 and 46%, respectively. We concluded that APN peptide II inhibits CNV size on days 7 and 14 by inhibiting the proliferation of endothelial cells in vivo and in vitro. APN peptide II may have therapeutic potential to inhibit CNV or wet AMD.
Asunto(s)
Adiponectina/uso terapéutico , Neovascularización Coroidal/tratamiento farmacológico , Fragmentos de Péptidos/uso terapéutico , Adiponectina/química , Secuencia de Aminoácidos , Animales , Neovascularización Coroidal/etiología , Neovascularización Coroidal/metabolismo , Células Endoteliales/metabolismo , Células Endoteliales/efectos de la radiación , Rayos Láser , Masculino , Ratones , Ratones Endogámicos C57BL , Modelos Animales , Fragmentos de Péptidos/química , Antígeno Nuclear de Célula en Proliferación/metabolismo , Antígeno Nuclear de Célula en Proliferación/fisiología , Estructura Terciaria de Proteína , Receptores de Adiponectina/metabolismo , Receptores de Adiponectina/fisiología , Factor A de Crecimiento Endotelial Vascular/metabolismo , Factor A de Crecimiento Endotelial Vascular/fisiologíaRESUMEN
The objective of the present study was to investigate the effect of alcohol and nicotine consumption on the pathogenesis of choroidal neovascularization (CNV) in rats after laser-photocoagulation. Confocal microscopic analysis demonstrated an increase in CNV complex size in rats fed with alcohol (2.3-fold), nicotine (1.9-fold), and the combination of alcohol and nicotine (2.7-fold) compared with the control groups. Immunohistochemical analysis revealed that alcohol and nicotine consumption increased MAC deposition and VEGF expression in laser spots. Expression of CD59 by RT-PCR and Western blot was drastically reduced in the animals that were fed with alcohol, nicotine and alcohol and nicotine compared to those fed with water alone and this was associated with exacerbation of CNV.
Asunto(s)
Consumo de Bebidas Alcohólicas/patología , Neovascularización Coroidal/patología , Etanol/toxicidad , Nicotina/toxicidad , Consumo de Bebidas Alcohólicas/metabolismo , Animales , Neovascularización Coroidal/metabolismo , Complejo de Ataque a Membrana del Sistema Complemento/metabolismo , Masculino , Ratas , Ratas Endogámicas BNRESUMEN
Evidence that poly(ADP-ribose) polymerase (PARP) activation plays an important role in diabetic complications is emerging. This study evaluated the role of PARP in rat and mouse models of advanced diabetic neuropathy. The orally active PARP inhibitor 10-(4-methylpiperazin-1-ylmethyl)-2H-7-oxa-1,2-diaza-benzo[de]anthracen-3-one (GPI-15427; formulated as a mesilate salt, 30 mg kg(-1) day(-1) in the drinking water for 10 weeks after the first 2 weeks without treatment) at least partially prevented PARP activation in peripheral nerve and DRG neurons, as well as thermal hypoalgesia, mechanical hyperalgesia, tactile allodynia, exaggerated response to formalin, and, most importantly, intraepidermal nerve fiber degeneration in streptozotocin-diabetic rats. These findings are consistent with the lack of small sensory nerve fiber dysfunction in diabetic PARP -/- mice. Furthermore, whereas diabetic PARP +/+ mice displayed approximately 46% intraepidermal nerve fiber loss, diabetic PARP -/- mice retained completely normal intraepidermal nerve fiber density. In conclusion, PARP activation is an important contributor to intraepidermal nerve fiber degeneration and functional changes associated with advanced Type 1 diabetic neuropathy. The results support a rationale for the development of potent and low-toxicity PARP inhibitors and PARP inhibitor-containing combination therapies.
Asunto(s)
Diabetes Mellitus Experimental/complicaciones , Neuropatías Diabéticas/prevención & control , Degeneración Nerviosa/prevención & control , Neuralgia/prevención & control , Nervios Periféricos/efectos de los fármacos , Inhibidores de Poli(ADP-Ribosa) Polimerasas , Animales , Neuropatías Diabéticas/etiología , Inhibidores Enzimáticos/uso terapéutico , Inmunohistoquímica , Masculino , Ratones , Degeneración Nerviosa/etiología , Neuralgia/etiología , Compuestos Orgánicos/uso terapéutico , Nervios Periféricos/patología , Poli(ADP-Ribosa) Polimerasas/efectos de los fármacos , Poli(ADP-Ribosa) Polimerasas/genética , Ratas , Ratas Wistar , Piel/inervaciónRESUMEN
Whereas the important role of free radicals in diabetes-associated complications is well established, the contributions of the highly reactive oxidant peroxynitrite have not been properly explored. The present study used a pharmacological approach to evaluate the role of peroxynitrite in peripheral diabetic neuropathy. Control and STZ-diabetic mice were maintained with or without treatment with the potent peroxynitrite decomposition catalyst Fe(III) tetramesitylporphyrin octasulfonate (FeTMPS), at doses of 5 or 10 mg/kg/day in the drinking water for 3 weeks after an initial 3 weeks without treatment. Mice with a 6-week duration of diabetes developed clearly manifest motor (MNCV) and sensory nerve conduction velocity (SNCV) deficits, thermal hypoalgesia (paw withdrawal, tail-flick, and hot plate tests), mechanical hypoalgesia (tail pressure Randall-Sellito test), tactile allodynia (flexible von Frey filament test), and approximately 44% loss of intraepidermal nerve fibers. They also had increased nitrotyrosine and poly(ADP-ribose) immunofluorescence in sciatic nerve, grey matter of the spinal cord, and dorsal root ganglion neurons. FeTMPS treatment alleviated or essentially corrected (at a dose of 10 mg/kg/day) MNCV and SNCV deficits, and was associated with less severe small sensory nerve fiber dysfunction and degeneration. Nitrotyrosine and poly(ADP-ribose) immunofluorescence in sciatic nerve, spinal cord, and dorsal root ganglion neurons in peroxynitrite decomposition catalyst-treated diabetic mice was markedly reduced. In conclusion, peroxynitrite contributes to large motor, large sensory, and small sensory fiber neuropathy in streptozotocin-diabetic mice. The findings provide rationale for development of potent peroxynitrite decomposition catalysts for the treatment of diabetic neuropathy.
Asunto(s)
Diabetes Mellitus Tipo 1/metabolismo , Neuropatías Diabéticas , Compuestos Férricos/metabolismo , Metaloporfirinas/metabolismo , Ácido Peroxinitroso/metabolismo , Animales , Conducta Animal/fisiología , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 1/complicaciones , Neuropatías Diabéticas/etiología , Neuropatías Diabéticas/metabolismo , Modelos Animales de Enfermedad , Compuestos Férricos/química , Humanos , Metaloporfirinas/química , Ratones , Ratones Endogámicos C57BL , Conducción Nerviosa , Estrés Oxidativo , Ácido Peroxinitroso/química , Poli(ADP-Ribosa) Polimerasas/metabolismo , Nervio Ciático/metabolismoRESUMEN
OBJECTIVE: Subjects with dietary obesity and pre-diabetes have an increased risk for developing both nerve conduction slowing and small sensory fiber neuropathy. Animal models of this type of neuropathy have not been described. This study evaluated neuropathic changes and their amenability to dietary and pharmacological interventions in mice fed a high-fat diet (HFD), a model of pre-diabetes and alimentary obesity. RESEARCH DESIGN AND METHODS: Female C57BL6/J mice were fed normal diets or HFDs for 16 weeks. RESULTS: HFD-fed mice developed obesity, increased plasma FFA and insulin concentrations, and impaired glucose tolerance. They also had motor and sensory nerve conduction deficits, tactile allodynia, and thermal hypoalgesia in the absence of intraepidermal nerve fiber loss or axonal atrophy. Despite the absence of overt hyperglycemia, the mice displayed augmented sorbitol pathway activity in the peripheral nerve, as well as 4-hydroxynonenal adduct nitrotyrosine and poly(ADP-ribose) accumulation and 12/15-lipoxygenase overexpression in peripheral nerve and dorsal root ganglion neurons. A 6-week feeding with normal chow after 16 weeks on HFD alleviated tactile allodynia and essentially corrected thermal hypoalgesia and sensory nerve conduction deficit without affecting motor nerve conduction slowing. Normal chow containing the aldose reductase inhibitor fidarestat (16 mg x kg(-1) x day (-1)) corrected all functional changes of HFD-induced neuropathy. CONCLUSIONS: Similar to human subjects with pre-diabetes and obesity, HFD-fed mice develop peripheral nerve functional, but not structural, abnormalities and, therefore, are a suitable model for evaluating dietary and pharmacological approaches to halt progression and reverse diabetic neuropathy at the earliest stage of the disease.
Asunto(s)
Aldehído Reductasa/antagonistas & inhibidores , Neuropatías Diabéticas/inducido químicamente , Grasas de la Dieta/farmacología , Imidazolidinas/uso terapéutico , Estado Prediabético/fisiopatología , Animales , Regulación de la Temperatura Corporal , Neuropatías Diabéticas/prevención & control , Modelos Animales de Enfermedad , Ácidos Grasos no Esterificados/sangre , Femenino , Ratones , Ratones Endogámicos C57BL , Neuronas Aferentes/fisiología , Obesidad/fisiopatología , Radioinmunoensayo , Umbral Sensorial , Tacto/fisiologíaRESUMEN
Whereas an important role of free radicals and oxidants in peripheral diabetic neuropathy is well established, the contribution of nitrosative stress and, in particular, of the highly reactive oxidant peroxynitrite, has not been properly explored. Our previous findings implicate peroxynitrite in diabetes-associated motor and sensory nerve conduction deficits and peripheral nerve energy deficiency and poly(ADP-ribose) polymerase activation associated with Type 1 diabetes. In this study the role of nitrosative stress in diabetic sensory neuropathy is evaluated. The peroxynitrite decomposition catalyst Fe(III) tetrakis-2-(N-triethylene glycol monomethyl ether)pyridyl porphyrin (FP15) was administered to control and streptozotocin (STZ)-diabetic mice at the dose of 5 mg kg(-1) day(-1) (FP15), for 3 weeks after initial 3 weeks without treatment. Mice with 6-week duration of diabetes developed clearly manifest thermal hypoalgesia (paw withdrawal, tail-flick, and hot plate tests), mechanical hypoalgesia (tail pressure Randall-Sellito test), tactile allodynia (flexible von Frey filament test), and approximately 38% loss of intraepidermal nerve fibers. They also had increased nitrotyrosine and poly(ADP-ribose) immunofluorescence in the sciatic nerve, grey matter of spinal cord, and dorsal root ganglion neurons. FP15 treatment was associated with alleviation of thermal and mechanical hypoalgesia. Tactile response threshold tended to increase in response to peroxynitrite decomposition catalyst treatment, but still remained approximately 59% lower compared with non-diabetic controls. Intraepidermal nerve fiber density was 25% higher in FP15-treated than in untreated diabetic rats, but the difference between two groups did not achieve statistical significance (p=0.054). Nitrotyrosine and poly(ADP-ribose) immunofluorescence in sciatic nerve, spinal cord, and dorsal root ganglion neurons of peroxynitrite decomposition catalyst-treated diabetic mice were markedly reduced. In conclusion, nitrosative stress plays an important role in sensory neuropathy associated with Type 1 diabetes. The findings provide rationale for further studies of peroxynitrite decomposition catalysts in a long-term diabetic model.
Asunto(s)
Diabetes Mellitus Experimental/complicaciones , Neuropatías Diabéticas/prevención & control , Metaloporfirinas/uso terapéutico , Ácido Peroxinitroso/metabolismo , Animales , Conducta Animal/efectos de los fármacos , Peso Corporal/efectos de los fármacos , Diabetes Mellitus Experimental/inducido químicamente , Diabetes Mellitus Experimental/fisiopatología , Neuropatías Diabéticas/etiología , Neuropatías Diabéticas/metabolismo , Técnica del Anticuerpo Fluorescente , Calor , Hiperalgesia/etiología , Hiperalgesia/prevención & control , Inmunohistoquímica , Masculino , Metaloporfirinas/metabolismo , Metaloporfirinas/farmacología , Ratones , Ratones Endogámicos C57BL , Fibras Nerviosas/efectos de los fármacos , Fibras Nerviosas/metabolismo , Fibras Nerviosas/patología , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Neuronas/patología , Umbral del Dolor/efectos de los fármacos , Poli Adenosina Difosfato Ribosa/metabolismo , Tiempo de Reacción/efectos de los fármacos , Nervio Ciático/efectos de los fármacos , Nervio Ciático/metabolismo , Nervio Ciático/patología , Raíces Nerviosas Espinales/efectos de los fármacos , Raíces Nerviosas Espinales/metabolismo , Raíces Nerviosas Espinales/patología , Estreptozocina , Tirosina/análogos & derivados , Tirosina/metabolismoRESUMEN
The aim of this study was to investigate the role of adiponectin (APN) in a mouse model of laser induced choroidal neovascularization (CNV). We have shown by immunohistochemistry that the expression of APN, adiponectin receptor 1, adiponectin receptor 2 and T cadherin gradually increased from day 1 to day 7 post-laser in laser treated mice compared to controls. Recombinant APN (rAPN) was injected intraperitoneally (i.p., 25 microg/mouse) or intravitreally (2 microg/eye) in lasered mice. Another set of lasered mice received APN peptide via i.p. (75 microg/mouse) or intravitreal (30 microg/eye) route. Control mice received a similar treatment with PBS, control protein or control peptide after laser treatment. We found that in the i.p. and intravitreal injection of rAPN resulted in 78% and 68% inhibition respectively in the size of CNV complex compared to control mice. Similar results were observed when APN peptide was injected intravitreally or i.p. Treatment with rAPN or the peptide resulted in decreased levels of vascular endothelial growth factor. Thus, APN inhibited choroidal angiogenesis and may have therapeutic implications in the treatment of wet age related macular degeneration.
Asunto(s)
Adiponectina/metabolismo , Coroides/irrigación sanguínea , Coroides/metabolismo , Neovascularización Coroidal/patología , Adiponectina/farmacología , Animales , Neovascularización Coroidal/inducido químicamente , Regulación de la Expresión Génica/efectos de los fármacos , Sustancias de Crecimiento/metabolismo , Rayos Láser , Ratones , Ratones Endogámicos C57BL , Modelos Moleculares , Péptidos/química , Péptidos/farmacología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas Recombinantes/farmacología , Factor A de Crecimiento Endotelial Vascular/genética , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Nitrosative stress contributes to nerve conduction slowing, thermal hypoalgesia, and impaired nitrergic innervation in animal models of Type 1 diabetes. The role for reactive nitrogen species in Type 2 diabetes-associated neuropathy remains unexplored. This study evaluated the role for nitrosative stress in functional and structural neuropathic changes in ob/ob mice, a model of Type 2 diabetes with mild hyperglycemia and obesity. Two structurally diverse peroxynitrite decomposition catalysts, Fe(III) tetrakis-2-(N-triethylene glycol monomethyl ether)-pyridyl porphyrin (FP15) and Fe(III) tetra-mesitylporphyrin octasulfonate (FeTMPS), were administered to control and 8-week-old ob/ob mice for 3 weeks at the doses of 5 mg kg(-1) day(-1) (FP15) and 5 and 10 mg kg(-1) day(-1) (FeTMPS). The 11-week-old ob/ob mice developed motor nerve conduction velocity (MNCV) and hind-limb digital sensory nerve conduction velocity (SNCV) deficits, thermal hypoalgesia, tactile allodynia, and a remarkable ( approximately 78%) loss of intraepidermal nerve fibers. They also had increased nitrotyrosine and poly(ADP-ribose) immunofluorescence in the sciatic nerve, spinal cord, and dorsal root ganglion neurons. Treatment with two structurally diverse peroxynitrite decomposition catalysts was associated with restoration of normal MNCV and SNCV, and alleviation of thermal hypoalgesia. Tactile response thresholds increased in response to peroxynitrite decomposition catalyst treatment, but still remained approximately 2.7- to 3.2-fold lower compared with non-diabetic controls. Intraepidermal nerve fiber loss was not alleviated by either FP15 or FeTMPS. Nitrotyrosine and poly(ADP-ribose) immunofluorescence in sciatic nerve, spinal cord, and dorsal root ganglia of peroxynitrite decomposition catalyst-treated ob/ob mice were essentially normal. In conclusion, nitrosative stress plays an important role in functional abnormalities associated with large motor, large sensory, and small sensory fiber neuropathy, but not in small sensory nerve fiber degeneration, in this animal model. Peroxynitrite decomposition catalysts alleviate Type 2 diabetes-associated sensory nerve dysfunction, likely by mechanism(s) not involving arrest of degenerative changes or enhanced regeneration of small sensory nerve fibers.
Asunto(s)
Nefropatías Diabéticas/patología , Leptina/deficiencia , Nitritos/metabolismo , Anestesia , Animales , Glucemia/metabolismo , Peso Corporal/fisiología , Diabetes Mellitus Tipo 2/patología , Diabetes Mellitus Tipo 2/fisiopatología , Nefropatías Diabéticas/fisiopatología , Inmunohistoquímica , Ratones , Ratones Endogámicos C57BL , Ratones Obesos , Neuronas Motoras/fisiología , Fibras Nerviosas/patología , Conducción Nerviosa/fisiología , Neuronas Aferentes/fisiología , Dolor/patología , Dimensión del Dolor , Ácido Peroxinitroso/metabolismo , Poli Adenosina Difosfato Ribosa/metabolismo , Tacto/fisiologíaRESUMEN
Whereas functional, metabolic, neurotrophic, and morphological abnormalities of peripheral diabetic neuropathy (PDN) have been extensively explored in streptozotocin-induced diabetic rats and mice (models of type 1 diabetes), insufficient information is available on manifestations and pathogenetic mechanisms of PDN in type 2 diabetic models. The latter could constitute a problem for clinical trial design because the vast majority of subjects with diabetes have type 2 (non-insulin dependent) diabetes. This study was aimed at characterization of PDN in leptin-deficient (ob/ob) mice, a model of type 2 diabetes with relatively mild hyperglycemia and obesity. ob/ob mice ( approximately 11 weeks old) clearly developed manifest sciatic motor nerve conduction velocity (MNCV) and hind-limb digital sensory nerve conduction velocity (SNCV) deficits, thermal hypoalgesia, tactile allodynia, and a remarkable ( approximately 78%) loss of intraepidermal nerve fibers. They also had increased sorbitol pathway activity in the sciatic nerve and increased nitrotyrosine and poly(ADP-ribose) immunofluorescence in the sciatic nerve, spinal cord, and dorsal root ganglion (DRG). Aldose reductase inhibition with fidarestat (16 mg . kg(-1) . d(-1)), administered to ob/ob mice for 6 weeks starting from 5 weeks of age, was associated with preservation of normal MNCV and SNCV and alleviation of thermal hypoalgesia and intraepidermal nerve fiber loss but not tactile allodynia. Sciatic nerve nitrotyrosine immunofluorescence and the number of poly(ADP-ribose)-positive nuclei in sciatic nerve, spinal cord, and DRGs of fidarestat-treated ob/ob mice did not differ from those in nondiabetic controls. In conclusion, the leptin-deficient ob/ob mouse is a new animal model that develops both large motor and sensory fiber and small sensory fiber PDN and responds to pathogenetic treatment. The results support the role for increased aldose reductase activity in functional and structural changes of PDN in type 2 diabetes.