RESUMO
Choroidal neovascularization (CNV) is the hallmark of wet age-related macular degeneration (AMD), a leading cause of irreversible blindness in the modern world. The objective for this study was to investigate the therapeutic potential of known antiangiogenic agents: thalidomide, senicapoc, and sodium butyrate. Dose-dependent effect of the agents on growth of ARPE-19 cells and human umbilical vein endothelial cells (HUVECs) was investigated with cell counting assays. Half-maximal inhibitory concentrations of thalidomide (765 µM and 1520 µM), senicapoc (50 µM and 79 µM), and sodium butyrate (933 µM and 557 µM) were determined for HUVECs and ARPE-19 cells, respectively. Immunofluorescence analysis showed decrease of VEGFA expression in both ARPE-19 cells and HUVECs after treatment only with thalidomide but not with senicapoc or sodium butyrate. Efficacy of the agents was studied in vivo with laser-induced CNV in C57BL/6 mice. Thalidomide (24 µg), senicapoc (4 µg), or sodium butyrate (100 µg) was intravitreally injected the day after CNV induction. Thalidomide, senicapoc, and sodium butyrate inhibited CNV size by 56%, 24%, and 21% respectively on day 7 post-laser. Thalidomide also reduced cobalt chloride induced increase of VEGFA mRNA in ARPE-19 (-33%) and protein in culture medium (-20%). Our results suggest that thalidomide may have more therapeutic potential than senicapoc or sodium butyrate for treatment of CNV or wet AMD.
Assuntos
Acetamidas/uso terapêutico , Inibidores da Angiogênese/uso terapêutico , Ácido Butírico/uso terapêutico , Neovascularização de Coroide/tratamento farmacológico , Talidomida/uso terapêutico , Compostos de Tritil/uso terapêutico , Acetamidas/farmacologia , Inibidores da Angiogênese/farmacologia , Animais , Ácido Butírico/farmacologia , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Neovascularização de Coroide/patologia , Modelos Animais de Doenças , Células Endoteliais da Veia Umbilical Humana , Humanos , Masculino , Camundongos Endogâmicos C57BL , Talidomida/farmacologia , Compostos de Tritil/farmacologiaRESUMO
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.
Assuntos
Modelos Animais de Doenças , Atrofia Geográfica/genética , Degeneração Macular/genética , Proteína Cofatora de Membrana/deficiência , Animais , Western Blotting , Feminino , Atrofia Geográfica/patologia , Degeneração Macular/patologia , Masculino , Proteína Cofatora de Membrana/genética , Camundongos , Camundongos KnockoutRESUMO
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.
Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Aspirina/farmacologia , Neovascularização de Coroide/tratamento farmacológico , Modelos Animais de Doenças , Degeneração Retiniana/tratamento farmacológico , Epitélio Pigmentado da Retina/efeitos dos fármacos , Animais , Linhagem Celular , Quimiocina CCL2/metabolismo , Neovascularização de Coroide/metabolismo , Neovascularização de Coroide/patologia , Dieta , Ensaio de Imunoadsorção Enzimática , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Degeneração Retiniana/metabolismo , Degeneração Retiniana/patologia , Epitélio Pigmentado da Retina/metabolismo , Epitélio Pigmentado da Retina/patologia , Fator A de Crescimento do Endotélio Vascular/metabolismo , Cicatrização/efeitos dos fármacosRESUMO
Dysregulation of the complement system is increasingly recognized as a contributing factor in age-related macular degeneration. Although the complement regulator CD46 is expressed ubiquitously in humans, in mouse it was previously thought to be expressed only on spermatozoa. We detected CD46 mRNA and protein in the posterior ocular segment (neuronal retina, retinal pigment epithelium, and choroid) of wild-type (WT) C57BL/6J mice. Cd46(-/-) knockout mice exhibited increased levels of the membrane attack complex and of vascular endothelial growth factor (VEGF) in the retina and choroid. The Cd46(-/-) mice were also more susceptible to laser-induced choroidal neovascularization (CNV). In Cd46(-/-) mice, 19% of laser spots were positive for CNV at day 2 after treatment, but no positive spots were detected in WT mice. At day 3, 42% of laser spots were positive in Cd46(-/-) mice, but only 11% in WT mice. A fully developed CNV complex was noted in both Cd46(-/-) and WT mice at day 7; however, lesion size was significantly (P < 0.05) increased in Cd46(-/-) mice. Our findings provide evidence for expression of CD46 in the mouse eye and a role for CD46 in protection against laser-induced CNV. We propose that the Cd46(-/-) mouse has a greater susceptibility to experimental CNV because of insufficient complement inhibition, which leads to increased membrane attack complex deposition and VEGF expression.
Assuntos
Neovascularização de Coroide/metabolismo , Proteína Cofatora de Membrana/metabolismo , Animais , Western Blotting , Ensaio de Imunoadsorção Enzimática , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
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.
Assuntos
Modelos Animais de Doenças , Atrofia Geográfica/patologia , Células Fotorreceptoras de Vertebrados/patologia , Polietilenoglicóis/toxicidade , Epitélio Pigmentado da Retina/patologia , Aminopeptidases/genética , Animais , Apoptose , Autofagia , Proteína Inibidora do Complemento C1/genética , Complemento C3/genética , Relação Dose-Resposta a Droga , Regulação da Expressão Gênica/fisiologia , Atrofia Geográfica/induzido quimicamente , Atrofia Geográfica/genética , Serina Peptidase 1 de Requerimento de Alta Temperatura A , Marcação In Situ das Extremidades Cortadas , Injeções Intraoculares , Masculino , Metaloproteinase 9 da Matriz/genética , Camundongos , Camundongos Endogâmicos C57BL , Serina Endopeptidases/genética , Fatores de TempoRESUMO
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.
Assuntos
Adiponectina/farmacologia , Corioide/irrigação sanguínea , Neovascularização de Coroide/prevenção & controle , Fragmentos de Peptídeos/farmacologia , Degeneração Macular Exsudativa/tratamento farmacológico , Adiponectina/uso terapêutico , Animais , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Corioide/efeitos dos fármacos , Corioide/patologia , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Células Endoteliais/fisiologia , Expressão Gênica/efeitos dos fármacos , Masculino , Fragmentos de Peptídeos/uso terapêutico , Antígeno Nuclear de Célula em Proliferação/metabolismo , Ratos Endogâmicos BN , Receptores de Adiponectina/metabolismo , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
The adaptor protein regulator for ubiquitous kinase/c-Cbl-interacting protein of 85kDa (Ruk/CIN85) was found to modulate HER1/EGFR signaling and processes like cell adhesion and apoptosis. Although these features imply a role in carcinogenesis, it is so far unknown how and by which molecular mechanisms Ruk/CIN85 could affect a certain tumor phenotype. By analyzing samples from breast cancer patients, we found high levels of Ruk(l)/CIN85 especially in lymph node metastases from patients with invasive breast adenocarcinomas, suggesting that Ruk(l)/CIN85 contributes to malignancy. Expression of Ruk(l)/CIN85 in weakly invasive breast adenocarcinoma cells deficient of Ruk(l)/CIN85 indeed converted them into more malignant cells. In particular, Ruk(l)/CIN85 reduced the growth rate, decreased cell adhesion, enhanced anchorage-independent growth, increased motility in both transwell migration and wound healing assays as well as affected the response to epidermal growth factor. Thereby, Ruk(l)/CIN85 led to a more rapid and prolonged epidermal growth factor-dependent activation of Src, Akt and ERK1/2 and treatment with the Src inhibitor PP2 and the PI3K inhibitor LY294002 abolished the Ruk(l)/CIN85-dependent changes in cell motility. Together, this study indicates that high levels of Ruk(l)/CIN85 contribute to the conversion of breast adenocarcinoma cells into a more malignant phenotype via modulation of the Src/Akt pathway.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Adesão Celular , Movimento Celular , Feminino , Humanos , Sistema de Sinalização das MAP Quinases , Proteína Oncogênica pp60(v-src)/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de SinaisRESUMO
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.
Assuntos
Quimiocina CCL2/biossíntese , Neovascularização de Coroide/metabolismo , Complexo de Ataque à Membrana do Sistema Complemento/metabolismo , Regulação da Expressão Gênica , Lasers/efeitos adversos , Fator A de Crescimento do Endotélio Vascular/biossíntese , Animais , Anticorpos Neutralizantes/farmacologia , Neovascularização de Coroide/etiologia , Neovascularização de Coroide/patologia , Modelos Animais de Doenças , Camundongos , Fatores de TempoRESUMO
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.
Assuntos
Neovascularização de Coroide/induzido quimicamente , Modelos Animais de Doenças , Portadores de Fármacos/efeitos adversos , Degeneração Macular , Polietilenoglicóis/efeitos adversos , Animais , Neovascularização de Coroide/metabolismo , Neovascularização de Coroide/patologia , Complemento C3/metabolismo , Complemento C9/metabolismo , Portadores de Fármacos/farmacologia , Camundongos , Polietilenoglicóis/farmacologia , Fatores de Tempo , Fator de Crescimento Transformador beta2/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
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.
Assuntos
Inibidores da Angiogênese/uso terapêutico , Anticorpos Monoclonais Humanizados/uso terapêutico , Neovascularização de Coroide/tratamento farmacológico , Nicotina/farmacologia , Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Adiponectina/uso terapêutico , Análise de Variância , Animais , Bevacizumab , Bungarotoxinas/farmacologia , Corioide/metabolismo , Neovascularização de Coroide/metabolismo , Neovascularização de Coroide/patologia , Modelos Animais de Doenças , Imuno-Histoquímica , Injeções Intravítreas , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Receptores Nicotínicos/metabolismo , Epitélio Pigmentado da Retina/metabolismo , Fatores de Crescimento do Endotélio Vascular/metabolismo , Receptor Nicotínico de Acetilcolina alfa7RESUMO
Age-related macular degeneration (AMD) is an eye disease that can cause central vision loss, particularly in the elderly population. There are 2 classes of AMD, wet-type and dry-type. Wet-type involves excess angiogenesis around the macula, referred to as choroidal neovascularization (CNV). This can result in leaky vessels, often causing more severe vision loss than dry-type AMD. Adiponectin peptide 1 (APNp1) has been shown to slow the progression of CNV. Here, we used a mouse model and FITC-labeled APNp1 to determine if APNp1 could be delivered effectively as an eye drop. Our experiment revealed that topically applied FITC-APNp1 could reach the macula of the eye, which is crucial for treating wet-type AMD. We also tested delivery of APNp1 via injection of an adeno-associated virus (AAV) vector in a mouse model of CNV. AAV is a harmless virus easy to manipulate and is very often used for protein or peptide deliveries. Results revealed an increase in the expression of APNp1 in the retina and choroid over a 28-day period. Finally, we investigated the mechanism by which APNp1 affects CNV by examining the expression of adiponectin receptor 1 (AdipoR1) and proliferating cell nuclear antigen (PCNA) in the retinal and choroidal tissue of the mouse eyes. AdipoR1 and PCNA were overexpressed in these tissues in mice with laser-induced CNV compared to naïve mice. Based on our data shown here, we think it will enhance our understanding of APNp1 as a therapeutic agent for wet-type AMD and possible treatment alternatives that could be more beneficial for patients.
Assuntos
Neovascularização de Coroide , Degeneração Macular Exsudativa , Adiponectina/genética , Idoso , Animais , Neovascularização de Coroide/genética , Neovascularização de Coroide/metabolismo , Modelos Animais de Doenças , Fluoresceína-5-Isotiocianato , Humanos , Camundongos , Soluções Oftálmicas , Peptídeos , Antígeno Nuclear de Célula em Proliferação/metabolismo , Receptores de Quinase C Ativada , Receptores de Adiponectina/genética , Receptores de Adiponectina/metabolismoRESUMO
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.
Assuntos
Antígenos CD59/metabolismo , Neovascularização de Coroide/metabolismo , Neovascularização Patológica/metabolismo , Proteínas Recombinantes/química , Animais , Apoptose , Membrana Celular/metabolismo , Proliferação de Células , Proteínas do Sistema Complemento/química , Imuno-Histoquímica , Inflamação , Degeneração Macular/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
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.
Assuntos
Neovascularização de Coroide/etiologia , Neovascularização de Coroide/metabolismo , Fator H do Complemento/fisiologia , Modelos Animais de Doenças , Lasers/efeitos adversos , Epitélio Pigmentado Ocular/metabolismo , Animais , Western Blotting , Neovascularização de Coroide/patologia , Fator H do Complemento/antagonistas & inibidores , Técnicas Imunoenzimáticas , Degeneração Macular/etiologia , Degeneração Macular/metabolismo , Degeneração Macular/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Epitélio Pigmentado Ocular/patologia , RNA Mensageiro/genética , RNA Interferente Pequeno/farmacologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Visão Ocular/fisiologiaRESUMO
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.
Assuntos
Mieloma Múltiplo , Proliferação de Células , Proteínas de Ligação a DNA , Humanos , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/genética , Complexo Repressor Polycomb 2 , Fatores de Transcrição/genéticaRESUMO
Primary plasma cell leukemia (pPCL) is a rare and aggressive form of multiple myeloma (MM) that is characterized by the presence of ≥20% circulating plasma cells. Overall survival remains poor despite advances of anti-MM therapy. The disease biology as well as molecular mechanisms that distinguish pPCL from non-pPCL MM remain poorly understood and, given the rarity of the disease, are challenging to study. In an attempt to identify key biological mechanisms that result in the aggressive pPCL phenotype, we performed whole-exome sequencing and gene expression analysis in 23 and 41 patients with newly diagnosed pPCL, respectively. The results reveal an enrichment of complex structural changes and high-risk mutational patterns in pPCL that explain, at least in part, the aggressive nature of the disease. In particular, pPCL patients with traditional low-risk features such as translocation t(11;14) or hyperdiploidy accumulated adverse risk genetic events that could account for the poor outcome in this group. Furthermore, gene expression profiling showed upregulation of adverse risk modifiers in pPCL compared to non-pPCL MM, while adhesion molecules and extracellular matrix proteins became increasingly downregulated. In conclusion, this is one of the largest studies to dissect pPCL on a genomic and molecular level.
Assuntos
Leucemia Plasmocitária/genética , Mutação , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Perfilação da Expressão Gênica , Regulação Leucêmica da Expressão Gênica , Genômica , Humanos , Leucemia Plasmocitária/patologia , Masculino , Pessoa de Meia-Idade , Taxa de Mutação , Transcriptoma , Translocação Genética , Sequenciamento do ExomaRESUMO
While the past decade has seen meaningful improvements in clinical outcomes for multiple myeloma patients, a subset of patients does not benefit from current therapeutics for unclear reasons. Many gene expression-based models of risk have been developed, but each model uses a different combination of genes and often involves assaying many genes making them difficult to implement. We organized the Multiple Myeloma DREAM Challenge, a crowdsourced effort to develop models of rapid progression in newly diagnosed myeloma patients and to benchmark these against previously published models. This effort lead to more robust predictors and found that incorporating specific demographic and clinical features improved gene expression-based models of high risk. Furthermore, post-challenge analysis identified a novel expression-based risk marker, PHF19, which has recently been found to have an important biological role in multiple myeloma. Lastly, we show that a simple four feature predictor composed of age, ISS, and expression of PHF19 and MMSET performs similarly to more complex models with many more gene expression features included.
Assuntos
Biomarcadores Tumorais/metabolismo , Ensaios Clínicos como Assunto/estatística & dados numéricos , Proteínas de Ligação a DNA/metabolismo , Epigênese Genética , Regulação Neoplásica da Expressão Gênica , Modelos Estatísticos , Mieloma Múltiplo/patologia , Fatores de Transcrição/metabolismo , Biomarcadores Tumorais/genética , Ciclo Celular , Proliferação de Células , Proteínas de Ligação a DNA/genética , Bases de Dados Factuais , Conjuntos de Dados como Assunto , Humanos , Mieloma Múltiplo/genética , Mieloma Múltiplo/metabolismo , Fatores de Transcrição/genética , Células Tumorais CultivadasRESUMO
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.
Assuntos
Adiponectina/uso terapêutico , Neovascularização de Coroide/tratamento farmacológico , Fragmentos de Peptídeos/uso terapêutico , Adiponectina/química , Sequência de Aminoácidos , Animais , Neovascularização de Coroide/etiologia , Neovascularização de Coroide/metabolismo , Células Endoteliais/metabolismo , Células Endoteliais/efeitos da radiação , Lasers , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Modelos Animais , Fragmentos de Peptídeos/química , Antígeno Nuclear de Célula em Proliferação/metabolismo , Antígeno Nuclear de Célula em Proliferação/fisiologia , Estrutura Terciária de Proteína , Receptores de Adiponectina/metabolismo , Receptores de Adiponectina/fisiologia , Fator A de Crescimento do Endotélio Vascular/metabolismo , Fator A de Crescimento do Endotélio Vascular/fisiologiaRESUMO
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.
Assuntos
Consumo de Bebidas Alcoólicas/patologia , Neovascularização de Coroide/patologia , Etanol/toxicidade , Nicotina/toxicidade , Consumo de Bebidas Alcoólicas/metabolismo , Animais , Neovascularização de Coroide/metabolismo , Complexo de Ataque à Membrana do Sistema Complemento/metabolismo , Masculino , Ratos , Ratos Endogâmicos BNRESUMO
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.
Assuntos
Diabetes Mellitus Experimental/complicações , Neuropatias Diabéticas/prevenção & controle , Degeneração Neural/prevenção & controle , Neuralgia/prevenção & controle , Nervos Periféricos/efeitos dos fármacos , Inibidores de Poli(ADP-Ribose) Polimerases , Animais , Neuropatias Diabéticas/etiologia , Inibidores Enzimáticos/uso terapêutico , Imuno-Histoquímica , Masculino , Camundongos , Degeneração Neural/etiologia , Neuralgia/etiologia , Compostos Orgânicos/uso terapêutico , Nervos Periféricos/patologia , Poli(ADP-Ribose) Polimerases/efeitos dos fármacos , Poli(ADP-Ribose) Polimerases/genética , Ratos , Ratos Wistar , Pele/inervaçãoRESUMO
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.