RESUMO
Degeneration of the retinal pigmented epithelium (RPE) and aberrant blood vessel growth in the eye are advanced-stage processes in blinding diseases such as age-related macular degeneration (AMD), which affect hundreds of millions of people worldwide. Loss of the RNase DICER1, an essential factor in micro-RNA biogenesis, is implicated in RPE atrophy. However, the functional implications of DICER1 loss in choroidal and retinal neovascularization are unknown. Here, we report that two independent hypomorphic mouse strains, as well as a separate model of postnatal RPE-specific DICER1 ablation, all presented with spontaneous RPE degeneration and choroidal and retinal neovascularization. DICER1 hypomorphic mice lacking critical inflammasome components or the innate immune adaptor MyD88 developed less severe RPE atrophy and pathological neovascularization. DICER1 abundance was also reduced in retinas of the JR5558 mouse model of spontaneous choroidal neovascularization. Finally, adenoassociated vector-mediated gene delivery of a truncated DICER1 variant (OptiDicer) reduced spontaneous choroidal neovascularization in JR5558 mice. Collectively, these findings significantly expand the repertoire of DICER1 in preserving retinal homeostasis by preventing both RPE degeneration and pathological neovascularization.
Assuntos
RNA Helicases DEAD-box/metabolismo , Degeneração Macular/metabolismo , Epitélio Pigmentado da Retina/irrigação sanguínea , Ribonuclease III/metabolismo , Animais , Neovascularização de Coroide/genética , Neovascularização de Coroide/metabolismo , Neovascularização de Coroide/patologia , Neovascularização de Coroide/fisiopatologia , RNA Helicases DEAD-box/genética , Humanos , Degeneração Macular/genética , Degeneração Macular/patologia , Degeneração Macular/fisiopatologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Degeneração Retiniana/genética , Degeneração Retiniana/metabolismo , Degeneração Retiniana/patologia , Degeneração Retiniana/fisiopatologia , Neovascularização Retiniana/genética , Neovascularização Retiniana/metabolismo , Neovascularização Retiniana/parasitologia , Neovascularização Retiniana/fisiopatologia , Epitélio Pigmentado da Retina/metabolismo , Epitélio Pigmentado da Retina/patologia , Ribonuclease III/genéticaRESUMO
PURPOSE: Chronic ocular pain is poorly understood and difficult to manage. We developed a murine model of corneal surface injury (CSI)-induced chronic ocular neuropathic pain. The study focuses on changes in corneal nerve morphology and associated short- and long-term pain-like behavior after CSI. METHODS: CSI was induced in mice by local application of an alkali solution (0.75 N NaOH). Corneal nerve architecture, morphology, density, and length were studied. Eye-wiping was evaluated before and after CSI in response to hypertonic saline (2 M NaCl). Naltrexone (NTX) or Naloxone-methiodide (NLX-me), opioid receptor antagonists, were given subcutaneously (s.c., 3 mg/kg) or topically (eye drop, 100 µM), and then an eye-wiping test was performed. RESULTS: CSI caused partial corneal deinnervation followed by gradual reinnervation. Regenerated nerves displayed increased tortuosity, beading, and branching. CSI enhanced hypertonic saline-induced eye-wiping behavior compared to baseline or sham-injury (P < 0.01). This hypersensitivity peaked at 10 days and subsided 14 days after CSI. Administration of NTX, or NLX-me, a selective peripheral opioid antagonist, reinstated eye-wiping behavior in the injury group, but not in the sham groups (P < 0.05). CONCLUSIONS: This study introduces a model of chronic ocular pain and corneal neuropathy following CSI. CSI induces central and peripheral opioid receptor-dependent latent sensitization (LS) that is unmasked by systemic or topical administration of opioid antagonists. TRANSLATIONAL RELEVANCE: This model of chronic ocular pain establishes LS as a new inhibitory mechanism in the oculotrigeminal system and may be used for potential diagnostic and therapeutic interventions for ocular neuropathy.
RESUMO
Optimal phototransduction requires separation of the avascular photoreceptor layer from the adjacent vascularized inner retina and choroid. Breakdown of peri-photoreceptor vascular demarcation leads to retinal angiomatous proliferation or choroidal neovascularization, two variants of vascular invasion of the photoreceptor layer in age-related macular degeneration (AMD), the leading cause of irreversible blindness in industrialized nations. Here we show that sFLT-1, an endogenous inhibitor of vascular endothelial growth factor A (VEGF-A), is synthesized by photoreceptors and retinal pigment epithelium (RPE), and is decreased in human AMD. Suppression of sFLT-1 by antibodies, adeno-associated virus-mediated RNA interference, or Cre/lox-mediated gene ablation either in the photoreceptor layer or RPE frees VEGF-A and abolishes photoreceptor avascularity. These findings help explain the vascular zoning of the retina, which is critical for vision, and advance two transgenic murine models of AMD with spontaneous vascular invasion early in life. DOI:http://dx.doi.org/10.7554/eLife.00324.001.
Assuntos
Neovascularização de Coroide/metabolismo , Degeneração Macular/metabolismo , Células Fotorreceptoras de Vertebrados/metabolismo , Neovascularização Retiniana/metabolismo , Epitélio Pigmentado da Retina/metabolismo , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Visão Ocular , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Anticorpos/farmacologia , Estudos de Casos e Controles , Neovascularização de Coroide/genética , Neovascularização de Coroide/patologia , Modelos Animais de Doenças , Regulação para Baixo , Feminino , Humanos , Degeneração Macular/genética , Degeneração Macular/patologia , Masculino , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células Fotorreceptoras de Vertebrados/patologia , Interferência de RNA , Neovascularização Retiniana/genética , Neovascularização Retiniana/patologia , Epitélio Pigmentado da Retina/patologia , Transdução de Sinais , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/deficiência , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/genéticaRESUMO
In this issue of Blood, Singh et al establish the existence of a new soluble isoform of vascular endothelial growth factor receptor 3 (sVEGFR-3), which is synthesized and secreted by corneal epithelial cells; they show that sVEGFR-3 modulates lymphangiogenesis by impounding vascular endothelial growth factor (VEGF) C and rendering it unable to activate its cognate receptors, thereby maintaining the natural alymphatic disposition of the cornea.
Assuntos
Córnea , Linfangiogênese/genética , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/fisiologia , Animais , HumanosRESUMO
Alu RNA accumulation due to DICER1 deficiency in the retinal pigmented epithelium (RPE) is implicated in geographic atrophy (GA), an advanced form of age-related macular degeneration that causes blindness in millions of individuals. The mechanism of Alu RNA-induced cytotoxicity is unknown. Here we show that DICER1 deficit or Alu RNA exposure activates the NLRP3 inflammasome and triggers TLR-independent MyD88 signaling via IL18 in the RPE. Genetic or pharmacological inhibition of inflammasome components (NLRP3, Pycard, Caspase-1), MyD88, or IL18 prevents RPE degeneration induced by DICER1 loss or Alu RNA exposure. These findings, coupled with our observation that human GA RPE contains elevated amounts of NLRP3, PYCARD, and IL18 and evidence of increased Caspase-1 and MyD88 activation, provide a rationale for targeting this pathway in GA. Our findings also reveal a function of the inflammasome outside the immune system and an immunomodulatory action of mobile elements.
Assuntos
Elementos Alu , RNA Helicases DEAD-box/metabolismo , Atrofia Geográfica/imunologia , Atrofia Geográfica/patologia , Inflamassomos/imunologia , Fator 88 de Diferenciação Mieloide/metabolismo , Epitélio Pigmentado da Retina/metabolismo , Ribonuclease III/metabolismo , Animais , Proteínas de Transporte/metabolismo , Atrofia Geográfica/metabolismo , Humanos , Inflamassomos/metabolismo , Camundongos , Proteína 3 que Contém Domínio de Pirina da Família NLR , Epitélio Pigmentado da Retina/patologia , Receptores Toll-Like/metabolismoRESUMO
The discovery of sequence-specific gene silencing by endogenous double-stranded RNAs (dsRNA) has propelled synthetic short-interfering RNAs (siRNAs) to the forefront of targeted pharmaceutical engineering. The first clinical trials utilized 21-nucleotide (nt) siRNAs for the treatment of neovascular age-related macular degeneration (AMD). Surprisingly, these compounds were not formulated for cell permeation, which is required for bona fide RNA interference (RNAi). We showed that these "naked" siRNAs suppress neovascularization in mice not via RNAi but via sequence-independent activation of cell surface Toll-like receptor-3 (TLR3). Here, we demonstrate that noninternalized siRNAs induce retinal degeneration in mice by activating surface TLR3 on retinal pigmented epithelial cells. Cholesterol conjugated siRNAs capable of cell permeation and triggering RNAi also induce the same phenotype. Retinal degeneration was not observed after treatment with siRNAs shorter than 21-nts. Other cytosolic dsRNA sensors are not critical to this response. TLR3 activation triggers caspase-3-mediated apoptotic death of the retinal pigment epithelium (RPE) via nuclear translocation of interferon regulatory factor-3. While this unexpected adverse effect of siRNAs has implications for future clinical trials, these findings also introduce a new preclinical model of geographic atrophy (GA), a late stage of dry AMD that causes blindness in millions worldwide.
Assuntos
Fator Regulador 3 de Interferon/metabolismo , RNA Interferente Pequeno/toxicidade , Degeneração Retiniana/induzido quimicamente , Receptor 3 Toll-Like/metabolismo , Animais , Caspase 3/metabolismo , Morte Celular/genética , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Ligação Proteica , RNA Interferente Pequeno/metabolismo , Degeneração Retiniana/metabolismo , Degeneração Retiniana/patologia , Epitélio Pigmentado da Retina/metabolismo , Transdução de SinaisRESUMO
Geographic atrophy (GA), an untreatable advanced form of age-related macular degeneration, results from retinal pigmented epithelium (RPE) cell degeneration. Here we show that the microRNA (miRNA)-processing enzyme DICER1 is reduced in the RPE of humans with GA, and that conditional ablation of Dicer1, but not seven other miRNA-processing enzymes, induces RPE degeneration in mice. DICER1 knockdown induces accumulation of Alu RNA in human RPE cells and Alu-like B1 and B2 RNAs in mouse RPE. Alu RNA is increased in the RPE of humans with GA, and this pathogenic RNA induces human RPE cytotoxicity and RPE degeneration in mice. Antisense oligonucleotides targeting Alu/B1/B2 RNAs prevent DICER1 depletion-induced RPE degeneration despite global miRNA downregulation. DICER1 degrades Alu RNA, and this digested Alu RNA cannot induce RPE degeneration in mice. These findings reveal a miRNA-independent cell survival function for DICER1 involving retrotransposon transcript degradation, show that Alu RNA can directly cause human pathology, and identify new targets for a major cause of blindness.
Assuntos
Elementos Alu/genética , RNA Helicases DEAD-box/deficiência , Degeneração Macular/genética , Degeneração Macular/patologia , RNA/genética , RNA/metabolismo , Ribonuclease III/deficiência , Animais , Morte Celular , Sobrevivência Celular , Células Cultivadas , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , Técnicas de Silenciamento de Genes , Humanos , Camundongos , MicroRNAs/metabolismo , Dados de Sequência Molecular , Oligonucleotídeos Antissenso , Fenótipo , Epitélio Pigmentado da Retina/enzimologia , Epitélio Pigmentado da Retina/metabolismo , Epitélio Pigmentado da Retina/patologia , Ribonuclease III/genética , Ribonuclease III/metabolismoRESUMO
BACKGROUND: Cancer metastasis contributes significantly to cancer mortality and is facilitated by lymphangiogenesis and angiogenesis. A new splicing variant, endogenous soluble vascular endothelial growth factor receptor-2 (esVEGFR-2) that we recently identified is an endogenous selective inhibitor of lymphangiogenesis. To evaluate the antimetastatic potential of esVEGFR-2, gene therapy with vector expressing esVEGFR-2 (pesVEGFR-2) or endostatin (pEndo) as a positive control was conducted on murine metastatic mammary cancer. METHODS: Syngeneic inoculated metastatic mammary cancers received direct intratumoral injection of pesVEGFR-2, pEndo or pVec as control, once a week for six weeks. In vivo gene electrotransfer was performed on the tumors after each injection. RESULTS: Deaths from metastasis were much lower in the pesVEGFR-2 and pEndo groups than in those of the pVec. Tumor volume was significantly lower in the pesVEGFR-2 and the pEndo groups throughout the study. Multiplicity of lymph node and lung metastatic nodules was significantly suppressed in the pesVEGFR-2 and pEndo groups. Moreover, the total number of overall metastasis including the other organs was also decreased in these groups. However, pesVEGFR-2 was not able to decrease the number of lungs, ovaries, kidneys and adrenals with metastasis as counted by unilateral or bilateral metastasis. The number of CD34+/Lyve-1â» blood microvessels was significantly decreased in the pEndo group, while the number of CD34â»/Lyve-1+ lymphatic vessels was significantly decreased in the pesVEGFR-2 and pEndo groups. In addition, a significant reduction in the number of dilated lymphatic vessels containing intraluminal cancer cells was observed in the pesVEGFR-2 and pEndo groups. Levels of apoptosis were significantly increased in the pEndo group, whereas the rates of cell proliferation were significantly decreased in the pesVEGFR-2 and pEndo groups. CONCLUSIONS: Our data demonstrate that esVEGFR-2 can inhibit mainly lymph node metastasis. The antimetastatic activity of esVEGFR-2 may be of high clinical significance in the treatment of metastatic breast cancer because lymph node involvement is a most important prognostic factor in cancer patients.
Assuntos
Linfangiogênese/efeitos dos fármacos , Metástase Linfática/genética , Neoplasias Mamárias Experimentais/tratamento farmacológico , Neoplasias Mamárias Experimentais/patologia , Receptores de Fatores de Crescimento do Endotélio Vascular/genética , Receptores de Fatores de Crescimento do Endotélio Vascular/uso terapêutico , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/uso terapêutico , Fatores de Crescimento do Endotélio Vascular/genética , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Feminino , Terapia Genética , Vetores Genéticos , Imunocompetência , Neoplasias Pulmonares/secundário , Linfangiogênese/genética , Neoplasias Mamárias Experimentais/genética , Camundongos , Camundongos Endogâmicos BALB C , Modelos Animais , Metástase Neoplásica , Isoformas de Proteínas , Receptores de Fatores de Crescimento do Endotélio Vascular/farmacologia , Carga Tumoral/efeitos dos fármacos , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/farmacologiaRESUMO
The vascular endothelial growth factor (VEGF) family of secreted proteins and their receptors are major regulators of blood vessel development (hemangiogenesis) and lymphatic vessel development (lymphangiogenesis). VEGF acts through a complex system of receptor tyrosine kinases, which can be membrane bound or soluble. New data concerning the receptor system are still emerging, thus contributing to the complexity of the system. Very recently a soluble form of VEGFR-2, termed sVEGFR-2, which is a result of alternative splicing, has been discovered. Earlier, it has been shown that a secreted/soluble form of VEGFR-1, termed sVEGFR-1, is produced by alternative splicing and exerts an antihemangiogenic effect by binding VEGF-A. The newly discovered spliced variant of sVEGFR-2 binds the lymphangiogenic growth factor VEGF-C and thus inhibits VEGF-C-induced activation of VEGFR-3, consequently inhibiting lymphatic endothelial cell proliferation. Its inactivation in murine embryos permits hyperplasia of dermal lymphatics and invasion of lymphatics into the cornea. Tumor lymphangiogenesis seems to influence the metastatic behavior of malignant cells. A correlation has been found between the downregulation of sVEGFR-2 and the malignant progression of neuroblastoma, which is characterized by lymphogenic metastases in progressed stages. Data show that lymphangiogenesis is regulated by both activators and inhibitors, and its balance is crucial in health and disease.
Assuntos
Linfangiogênese/fisiologia , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/fisiologia , Animais , Humanos , Camundongos , Ligação Proteica , Solubilidade , Fator A de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
PURPOSE: Tumor progression correlates with the induction of a dense supply of blood vessels and the formation of peritumoral lymphatics. Hemangiogenesis and lymphangiogenesis are potently regulated by members of the vascular endothelial growth factor (VEGF) family. Previous studies have indicated the upregulation of VEGF-A and -C in progressed neuroblastoma, however, quantification was performed using semiquantitative methods, or patients who had received radiotherapy or chemotherapy were studied. EXPERIMENTAL DESIGN: We have analyzed primary neuroblastoma from 49 patients using real-time reverse transcription-PCR and quantified VEGF-A, -C, and -D and VEGF receptors (VEGFR)-1, 2, 3, as well as the soluble form of VEGFR2 (sVEGFR-2), which has recently been characterized as an endogenous inhibitor of lymphangiogenesis. None of the patients had received radiotherapy or chemotherapy before tumor resection. RESULTS: We did not observe upregulation of VEGF-A, -C, and -D in metastatic neuroblastoma, but found significant downregulation of the lymphangiogenesis inhibitor sVEGFR-2 in metastatic stages III, IV, and IVs. In stage IV neuroblastoma, there were tendencies for the upregulation of VEGF-A and -D and the downregulation of the hemangiogenesis/lymphangiogenesis inhibitors VEGFR-1 and sVEGFR-2 in MYCN-amplified tumors. Similarly, MYCN transfection of the neuroblastoma cell line SH-EP induced the upregulation of VEGF-A and -D and the switching-off of sVEGFR-2. CONCLUSION: We provide evidence for the downregulation of the lymphangiogenesis inhibitor sVEGFR-2 in metastatic neuroblastoma stages, which may promote lymphogenic metastases. Downregulation of hemangiogenesis and lymphangiogenesis inhibitors VEGFR-1 and sVEGFR-2, and upregulation of angiogenic activators VEGF-A and VEGF-D in MYCN-amplified stage IV neuroblastoma supports the crucial effect of this oncogene on neuroblastoma progression.
Assuntos
Linfangiogênese/genética , Neuroblastoma/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Progressão da Doença , Regulação para Baixo , Ensaio de Imunoadsorção Enzimática , Humanos , Proteína Proto-Oncogênica N-Myc , Invasividade Neoplásica/genética , Estadiamento de Neoplasias , Neuroblastoma/genética , Neuroblastoma/patologia , Proteínas Nucleares/genética , Proteínas Oncogênicas/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo , Fator C de Crescimento do Endotélio Vascular/genética , Fator C de Crescimento do Endotélio Vascular/metabolismo , Fator D de Crescimento do Endotélio Vascular/genética , Fator D de Crescimento do Endotélio Vascular/metabolismo , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/genética , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/genética , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
Disruption of the precise balance of positive and negative molecular regulators of blood and lymphatic vessel growth can lead to myriad diseases. Although dozens of natural inhibitors of hemangiogenesis have been identified, an endogenous selective inhibitor of lymphatic vessel growth has not to our knowledge been previously described. We report the existence of a splice variant of the gene encoding vascular endothelial growth factor receptor-2 (Vegfr-2) that encodes a secreted form of the protein, designated soluble Vegfr-2 (sVegfr-2), that inhibits developmental and reparative lymphangiogenesis by blocking Vegf-c function. Tissue-specific loss of sVegfr-2 in mice induced, at birth, spontaneous lymphatic invasion of the normally alymphatic cornea and hyperplasia of skin lymphatics without affecting blood vasculature. Administration of sVegfr-2 inhibited lymphangiogenesis but not hemangiogenesis induced by corneal suture injury or transplantation, enhanced corneal allograft survival and suppressed lymphangioma cellular proliferation. Naturally occurring sVegfr-2 thus acts as a molecular uncoupler of blood and lymphatic vessels; modulation of sVegfr-2 might have therapeutic effects in treating lymphatic vascular malformations, transplantation rejection and, potentially, tumor lymphangiogenesis and lymphedema (pages 993-994).
Assuntos
Linfangiogênese/genética , Linfangiogênese/fisiologia , Vasos Linfáticos/fisiologia , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/fisiologia , Processamento Alternativo , Animais , Animais Recém-Nascidos , Sequência de Bases , Córnea/irrigação sanguínea , Córnea/crescimento & desenvolvimento , Córnea/metabolismo , DNA Complementar/genética , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Dados de Sequência Molecular , Fator C de Crescimento do Endotélio Vascular/antagonistas & inibidores , Fator C de Crescimento do Endotélio Vascular/fisiologia , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/deficiênciaRESUMO
Age-related macular degeneration (AMD), a leading cause of blindness worldwide, is as prevalent as cancer in industrialized nations. Most blindness in AMD results from invasion of the retina by choroidal neovascularisation (CNV). Here we show that the eosinophil/mast cell chemokine receptor CCR3 is specifically expressed in choroidal neovascular endothelial cells in humans with AMD, and that despite the expression of its ligands eotaxin-1, -2 and -3, neither eosinophils nor mast cells are present in human CNV. Genetic or pharmacological targeting of CCR3 or eotaxins inhibited injury-induced CNV in mice. CNV suppression by CCR3 blockade was due to direct inhibition of endothelial cell proliferation, and was uncoupled from inflammation because it occurred in mice lacking eosinophils or mast cells, and was independent of macrophage and neutrophil recruitment. CCR3 blockade was more effective at reducing CNV than vascular endothelial growth factor A (VEGF-A) neutralization, which is in clinical use at present, and, unlike VEGF-A blockade, is not toxic to the mouse retina. In vivo imaging with CCR3-targeting quantum dots located spontaneous CNV invisible to standard fluorescein angiography in mice before retinal invasion. CCR3 targeting might reduce vision loss due to AMD through early detection and therapeutic angioinhibition.
Assuntos
Degeneração Macular/diagnóstico , Degeneração Macular/terapia , Receptores CCR3/antagonistas & inibidores , Receptores CCR3/metabolismo , Animais , Movimento Celular , Proliferação de Células , Células Cultivadas , Quimiocina CCL11/antagonistas & inibidores , Quimiocina CCL11/metabolismo , Quimiocina CCL24/antagonistas & inibidores , Quimiocina CCL24/metabolismo , Quimiocina CCL26 , Quimiocinas CC/antagonistas & inibidores , Quimiocinas CC/metabolismo , Corioide/irrigação sanguínea , Corioide/citologia , Corioide/metabolismo , Neovascularização de Coroide/diagnóstico , Neovascularização de Coroide/metabolismo , Modelos Animais de Doenças , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Humanos , Inflamação , Leucócitos , Ligantes , Degeneração Macular/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Pontos Quânticos , Receptores CCR3/análise , Receptores CCR3/genética , Receptores CCR3/imunologia , Retina/efeitos dos fármacos , Retina/patologia , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Fator A de Crescimento do Endotélio Vascular/imunologiaRESUMO
Neovascularization in response to tissue injury consists of the dual invasion of blood (hemangiogenesis) and lymphatic (lymphangiogenesis) vessels. We reported recently that 21-nt or longer small interfering RNAs (siRNAs) can suppress hemangiogenesis in mouse models of choroidal neovascularization and dermal wound healing independently of RNA interference by directly activating Toll-like receptor 3 (TLR3), a double-stranded RNA immune receptor, on the cell surface of blood endothelial cells. Here, we show that a 21-nt nontargeted siRNA suppresses both hemangiogenesis and lymphangiogenesis in mouse models of neovascularization induced by corneal sutures or hindlimb ischemia as efficiently as a 21-nt siRNA targeting vascular endothelial growth factor-A. In contrast, a 7-nt nontargeted siRNA, which is too short to activate TLR3, does not block hemangiogenesis or lymphangiogenesis in these models. Exposure to 21-nt siRNA, which we demonstrate is not internalized unless cell-permeating moieties are used, triggers phosphorylation of cell surface TLR3 on lymphatic endothelial cells and induces apoptosis. These findings introduce TLR3 activation as a method of jointly suppressing blood and lymphatic neovascularization and simultaneously raise new concerns about the undesirable effects of siRNAs on both circulatory systems.
Assuntos
Vasos Linfáticos/metabolismo , Neovascularização Fisiológica , RNA Interferente Pequeno/genética , Receptor 3 Toll-Like/metabolismo , Animais , Apoptose , Proliferação de Células , Células Endoteliais/citologia , Membro Posterior/irrigação sanguínea , Membro Posterior/metabolismo , Camundongos , Fosforilação , Receptor 3 Toll-Like/genéticaRESUMO
Vascular endothelial growth factor receptor-1 (VEGFR-1, also known as Flt-1) is involved in complex biological processes often associated to severe pathological conditions like cancer, inflammation, and metastasis formation. Consequently, the search for antagonists of Flt-1 has recently gained a growing interest. Here we report the identification of a tetrameric tripeptide from a combinatorial peptide library built using non-natural amino acids, which binds Flt-1 and inhibits in vitro its interaction with placental growth factor (PlGF) and vascular endothelial growth factor (VEGF) A and B (IC(50) approximately 10 microm). The peptide is stable in serum for 7 days and prevents both Flt-1 phosphorylation and the capillary-like tube formation of human primary endothelial cells stimulated by PlGF or VEGF-A. Conversely, the identified peptide does not interfere in VEGF-induced VEGFR-2 activation. In vivo, this peptide inhibits VEGF-A- and PlGF-induced neoangiogenesis in the chicken embryo chorioallantoic membrane assay. In contrast, in the cornea, where avascularity is maintained by high levels of expression of the soluble form of Flt-1 receptor (sFlt-1) that prevents the VEGF-A activity, the peptide is able to stimulate corneal mouse neovascularization in physiological condition, as reported previously for others neutralizing anti-Flt-1 molecules. This tetrameric tripeptide represents a new, promising compound for therapeutic approaches in pathologies where Flt-1 activation plays a crucial role.
Assuntos
Regulação da Expressão Gênica , Neovascularização Patológica , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Fator A de Crescimento do Endotélio Vascular/metabolismo , Animais , Embrião de Galinha , Membrana Corioalantoide/química , Técnicas de Química Combinatória , Córnea/metabolismo , Humanos , Concentração Inibidora 50 , Camundongos , Camundongos Endogâmicos BALB C , Neovascularização Fisiológica , Peptídeos/química , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/química , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
Clinical trials of small interfering RNA (siRNA) targeting vascular endothelial growth factor-A (VEGFA) or its receptor VEGFR1 (also called FLT1), in patients with blinding choroidal neovascularization (CNV) from age-related macular degeneration, are premised on gene silencing by means of intracellular RNA interference (RNAi). We show instead that CNV inhibition is a siRNA-class effect: 21-nucleotide or longer siRNAs targeting non-mammalian genes, non-expressed genes, non-genomic sequences, pro- and anti-angiogenic genes, and RNAi-incompetent siRNAs all suppressed CNV in mice comparably to siRNAs targeting Vegfa or Vegfr1 without off-target RNAi or interferon-alpha/beta activation. Non-targeted (against non-mammalian genes) and targeted (against Vegfa or Vegfr1) siRNA suppressed CNV via cell-surface toll-like receptor 3 (TLR3), its adaptor TRIF, and induction of interferon-gamma and interleukin-12. Non-targeted siRNA suppressed dermal neovascularization in mice as effectively as Vegfa siRNA. siRNA-induced inhibition of neovascularization required a minimum length of 21 nucleotides, a bridging necessity in a modelled 2:1 TLR3-RNA complex. Choroidal endothelial cells from people expressing the TLR3 coding variant 412FF were refractory to extracellular siRNA-induced cytotoxicity, facilitating individualized pharmacogenetic therapy. Multiple human endothelial cell types expressed surface TLR3, indicating that generic siRNAs might treat angiogenic disorders that affect 8% of the world's population, and that siRNAs might induce unanticipated vascular or immune effects.
Assuntos
Terapia Genética/métodos , Imunidade Inata/imunologia , Neovascularização Patológica/imunologia , Neovascularização Patológica/prevenção & controle , RNA Interferente Pequeno/imunologia , RNA Interferente Pequeno/metabolismo , Receptor 3 Toll-Like/metabolismo , Animais , Linhagem Celular , Células Endoteliais/metabolismo , Humanos , Interferon gama/imunologia , Interleucina-12/imunologia , Degeneração Macular/complicações , Degeneração Macular/genética , Degeneração Macular/terapia , Camundongos , Camundongos Endogâmicos C57BL , Neovascularização Patológica/genética , Neovascularização Patológica/terapia , RNA Interferente Pequeno/química , RNA Interferente Pequeno/genética , Receptor 3 Toll-Like/química , Receptor 3 Toll-Like/genética , Fator A de Crescimento do Endotélio Vascular/genéticaRESUMO
OBJECTIVE: The aim of this study was to observe areas of brain activation with painful hot stimulation to the trigeminal nerve. STUDY DESIGN: Nine healthy pain-free women (mean age 26.2 +/- 6.9 yrs) with a natural, regular menstrual cycle participated in the study. Whole-brain functional magnetic resonance imaging (fMRI) data were acquired for each participant on day 2 or 3 after the onset of menses using echo-planar imaging at 1.5T with near-isotropic spatial resolution and a temporal resolution of 4 s. RESULTS: Whole-brain fMRI with a Peltier thermode inside the head coil yielded a feasible imaging protocol with little disturbance from the thermode. Painful thermal stimulation of the left trigeminal system activated discrete brain regions within the insula, cingulate gyrus, thalamus, inferior parietal lobe/postcentral gyrus, right middle and inferior frontal gyri, cuneus, precuneus, and precentral gyrus. CONCLUSION: Painful stimulation of the trigeminal nerve resulted in activation of similar brain areas generally known for pain processing of painful peripheral stimulation.
Assuntos
Mapeamento Encefálico/métodos , Cerebelo/fisiologia , Córtex Cerebral/fisiologia , Dor/fisiopatologia , Tálamo/fisiologia , Nervo Trigêmeo/fisiopatologia , Adulto , Feminino , Temperatura Alta , Humanos , Imageamento por Ressonância Magnética/métodos , Limiar da Dor/fisiologia , Estimulação FísicaRESUMO
Corneal avascularity-the absence of blood vessels in the cornea-is required for optical clarity and optimal vision, and has led to the cornea being widely used for validating pro- and anti-angiogenic therapeutic strategies for many disorders. But the molecular underpinnings of the avascular phenotype have until now remained obscure and are all the more remarkable given the presence in the cornea of vascular endothelial growth factor (VEGF)-A, a potent stimulator of angiogenesis, and the proximity of the cornea to vascularized tissues. Here we show that the cornea expresses soluble VEGF receptor-1 (sVEGFR-1; also known as sflt-1) and that suppression of this endogenous VEGF-A trap by neutralizing antibodies, RNA interference or Cre-lox-mediated gene disruption abolishes corneal avascularity in mice. The spontaneously vascularized corneas of corn1 and Pax6+/- mice and Pax6+/- patients with aniridia are deficient in sflt-1, and recombinant sflt-1 administration restores corneal avascularity in corn1 and Pax6+/- mice. Manatees, the only known creatures uniformly to have vascularized corneas, do not express sflt-1, whereas the avascular corneas of dugongs, also members of the order Sirenia, elephants, the closest extant terrestrial phylogenetic relatives of manatees, and other marine mammals (dolphins and whales) contain sflt-1, indicating that it has a crucial, evolutionarily conserved role. The recognition that sflt-1 is essential for preserving the avascular ambit of the cornea can rationally guide its use as a platform for angiogenic modulators, supports its use in treating neovascular diseases, and might provide insight into the immunological privilege of the cornea.
Assuntos
Córnea/irrigação sanguínea , Córnea/metabolismo , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Animais , Deleção de Genes , Camundongos , Neovascularização Fisiológica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Solubilidade , Trichechus , Fator A de Crescimento do Endotélio Vascular/metabolismo , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/deficiência , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/genéticaRESUMO
The pathophysiology of burning mouth disorder (BMD) is not clearly understood, but central neuropathic mechanisms are thought to be involved. The aim of this study was to gain insight into the pathophysiology associated with BMD by using functional magnetic resonance imaging (fMRI). Areas of brain activation following thermal stimulation of the trigeminal nerve of eight female patients with BMD (mean age 49.1+/-10.1) were mapped using fMRI and compared with those of eight matched pain-free volunteers (mean age 50.3+/-12.3). Qualitative and quantitative differences in brain activation patterns between the two study groups were demonstrated. BMD patients displayed greater fractional signal changes in the right anterior cingulate cortex (BA 32/24) and bilateral precuneus than did controls (p<0.005). The control group showed larger fractional signal changes in the bilateral thalamus, right middle frontal gyrus, right pre-central gyrus, left lingual gyrus, and cerebellum than did the BMD patients (p<0.005). In addition, BMD patients had less volumetric activation throughout the entire brain compared to the control group. Overall, BMD patients displayed brain activation patterns similar to those of patients with other neuropathic pain conditions and appear to process thermal painful stimulation to the trigeminal nerve qualitatively and quantitatively different than pain-free individuals. These findings suggest that brain hypoactivity may be an important feature in the pathophysiology of BMD.
Assuntos
Encéfalo/fisiopatologia , Síndrome da Ardência Bucal/fisiopatologia , Temperatura Alta , Imageamento por Ressonância Magnética , Adulto , Comportamento , Encéfalo/patologia , Mapeamento Encefálico , Síndrome da Ardência Bucal/diagnóstico , Síndrome da Ardência Bucal/psicologia , Estudos de Casos e Controles , Feminino , Giro do Cíngulo/fisiopatologia , Humanos , Pessoa de Meia-Idade , Estimulação Física , Testes Psicológicos , Tálamo/fisiopatologia , Nervo Trigêmeo/fisiopatologiaRESUMO
PURPOSE: Several studies have shown that women express higher pain sensitivity during periods of low estrogen than during periods of high estrogen. The aim of this study was to show whether the difference in pain sensitivity could be visualized as a function of brain activity by means of functional magnetic resonance imaging (fMRI). METHODS: Nine healthy, pain-free women (mean age, 26.2 +/- 6.9 years) with a natural, regular menstrual cycle participated in the study. Whole-brain fMRI data were acquired during a period of high and during a period of low estrogen at 1.5 T using echo-planar imaging with near-isotropic spatial resolution and a temporal resolution of 4 seconds. Heat pain thresholds were obtained before the scans, and pain ratings were obtained before and after each scan. Blood samples were taken after each scan to verify the appropriate level of estrogen. RESULTS: The heat pain thresholds during the low (46.4 degrees +/- 3.5 degrees C) and high (46.4 degrees +/- 3.8 degrees C) estrogen conditions were not significantly different. The pain ratings before (4.6 +/- 2.2 low versus 3.6 +/- 2.1 high) and during the scans (4.4 +/- 2.4 low versus 4.7 +/- 2.3 high) also did not differ between the 2 conditions. Generally, similar patterns of activation were observed for both estrogen conditions. However, significant differences were found in the magnitude of activation of the anterior part of the anterior cingulate (BA 24/32), the cerebellum, and the precuneus. Furthermore, activations in the anterior part of the anterior cingulate, left cerebellum, and precuneus were unique to the low-estrogen phase. These regions have been linked with attention to or anticipation of pain. CONCLUSIONS: The results of this study suggest that the affective component of pain may be enhanced during the low-estrogen phase of the menstrual cycle in healthy women.
Assuntos
Encéfalo/fisiologia , Estrogênios/sangue , Temperatura Alta , Ciclo Menstrual/sangue , Limiar da Dor/fisiologia , Adulto , Cerebelo/fisiologia , Córtex Cerebral/fisiologia , Estrogênios/fisiologia , Feminino , Humanos , Imageamento por Ressonância Magnética , Ciclo Menstrual/fisiologia , Dor/sangue , Dor/fisiopatologia , Medição da Dor , Estimulação Física/métodosRESUMO
This study was designed to assess whether the number and type of medical conditions elicited by self-report on a questionnaire were equivalent to those obtained through verbal inquiry by blinded trained dentists. The study sample consisted of 100 adult patients who were seeking treatment from the University of Kentucky Orofacial Pain Center. Evaluations occurred between September 2003 and January 2004. Patients completed a standardized medical health questionnaire containing 92 questions. Subsequently, the patients were questioned verbally about their medical history by uniformly trained dentists, using a systems review approach as part of routine clinical protocol. The medical histories obtained by both methods were statistically analyzed using Kappa values, paired and independent sample t-tests. The nature of the questionnaire did not distinguish between past and present conditions, and did not give information regarding onset, severity, duration, and impact of the marked conditions. It was concluded that even though a self-reported health questionnaire is precise and consistent, more detailed information can be obtained by verifying marked medical conditions through a verbal inquiry. Every history and examination should include a combination of both a self-reported medical health questionnaire and a verbal inquiry to aid in diagnosis and treatment.