RESUMEN
TAR DNA-binding protein 43 (TDP-43) is a DNA/RNA-binding protein that regulates gene expression, and its malfunction in neurons has been causally associated with multiple neurodegenerative disorders. Although progress has been made in understanding the functions of TDP-43 in neurons, little is known about its roles in endothelial cells (ECs), angiogenesis, and vascular function. Using inducible EC-specific TDP-43-KO mice, we showed that TDP-43 is required for sprouting angiogenesis, vascular barrier integrity, and blood vessel stability. Postnatal EC-specific deletion of TDP-43 led to retinal hypovascularization due to defects in vessel sprouting associated with reduced EC proliferation and migration. In mature blood vessels, loss of TDP-43 disrupted the blood-brain barrier and triggered vascular degeneration. These vascular defects were associated with an inflammatory response in the CNS with activation of microglia and astrocytes. Mechanistically, deletion of TDP-43 disrupted the fibronectin matrix around sprouting vessels and reduced ß-catenin signaling in ECs. Together, our results indicate that TDP-43 is essential for the formation of a stable and mature vasculature.
Asunto(s)
Células Endoteliales , Enfermedades Neuroinflamatorias , Ratones , Animales , Células Endoteliales/metabolismo , Angiogénesis , Neovascularización Fisiológica/genética , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismoRESUMEN
Pericytes are known as the mural cells in small-caliber vessels that interact closely with the endothelium. Pericytes play a key role in vasculature formation and homeostasis, and when dysfunctional contribute to vasculature-related diseases such as diabetic retinopathy and neurodegenerative conditions. In addition, significant extravascular roles of pathological pericytes are being discovered with relevant implications for cancer and fibrosis. Pericyte research is challenged by the lack of consistent molecular markers and clear discrimination criteria versus other (mural) cells. However, advances in single-cell approaches are uncovering and clarifying mural cell identities, biological functions, and ontogeny across organs. We discuss the latest developments in pericyte pathobiology to inform future research directions and potential outcomes.
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Endotelio Vascular , Pericitos , Humanos , Pericitos/fisiología , Biomarcadores , HomeostasisRESUMEN
Phosphoinositide 3-kinases (PI3Ks) phosphorylate intracellular inositol lipids to regulate signaling and intracellular vesicular trafficking. Mammals have eight PI3K isoforms, of which class I PI3Kα and class II PI3K-C2α are essential for vascular development. The class II PI3K-C2ß is also abundant in endothelial cells. Using in vivo and in vitro approaches, we found that PI3K-C2ß was a critical regulator of blood vessel growth by restricting endothelial mTORC1 signaling. Mice expressing a kinase-inactive form of PI3K-C2ß displayed enlarged blood vessels without corresponding changes in endothelial cell proliferation or migration. Instead, inactivation of PI3K-C2ß resulted in an increase in the size of endothelial cells, particularly in the sprouting zone of angiogenesis. Mechanistically, we showed that the aberrantly large size of PI3K-C2ß mutant endothelial cells was caused by mTORC1 activation, which sustained growth in these cells. Consistently, pharmacological inhibition of mTORC1 with rapamycin normalized vascular morphogenesis in PI3K-C2ß mutant mice. Together, these results identify PI3K-C2ß as a crucial determinant of endothelial signaling and illustrate the importance of mTORC1 regulation during angiogenic growth.
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Células Endoteliales , Fosfatidilinositol 3-Quinasas , Animales , Ratones , Proliferación Celular , Células Endoteliales/metabolismo , Mamíferos/metabolismo , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Isoformas de Proteínas , Transducción de SeñalRESUMEN
To evaluate a new animal model of chronic glaucoma induced using a single injection of fibronectin-loaded biodegradable PLGA microspheres (Ms) to test prolonged therapies. 30 rats received a single injection of fibronectin-PLGA-Ms suspension (MsF) in the right eye, 10 received non-loaded PLGA-Ms suspension (Control), and 17 were non-injected (Healthy). Follow-up was performed (24 weeks), evaluating intraocular pressure (IOP), optical coherence tomography (OCT), histology and electroretinography. The right eyes underwent a progressive increase in IOP, but only induced cohorts reached hypertensive values. The three cohorts presented a progressive decrease in ganglion cell layer (GCL) thickness, corroborating physiological age-related loss of ganglion cells. Injected cohorts (MsF > Control) presented greater final GCL thickness. Histological exams explain this paradox: the MsF cohort showed lower ganglion cell counts but higher astrogliosis and immune response. A sequential trend of functional damage was recorded using scotopic electroretinography (MsF > Control > Healthy). It seems to be a function-structure correlation: in significant astrogliosis, early functional damage can be detected by electroretinography, and structural damage can be detected by histological exams but not by OCT. Males presented higher IOP and retinal and GCL thicknesses and lower electroretinography. A minimally invasive chronic glaucoma model was induced by a single injection of biodegradable Ms.
Asunto(s)
Glaucoma , Presión Intraocular , Humanos , Masculino , Animales , Ratas , Fibronectinas , Gliosis , Microesferas , Glaucoma/tratamiento farmacológico , RetinaRESUMEN
Reciprocal interactions between endothelial cells (ECs) and adipocytes are fundamental to maintain white adipose tissue (WAT) homeostasis, as illustrated by the activation of angiogenesis upon WAT expansion, a process that is impaired in obesity. However, the molecular mechanisms underlying the crosstalk between ECs and adipocytes remain poorly understood. Here, we show that local production of polyamines in ECs stimulates adipocyte lipolysis and regulates WAT homeostasis in mice. We promote enhanced cell-autonomous angiogenesis by deleting Pten in the murine endothelium. Endothelial Pten loss leads to a WAT-selective phenotype, characterized by reduced body weight and adiposity in pathophysiological conditions. This phenotype stems from enhanced fatty acid ß-oxidation in ECs concomitant with a paracrine lipolytic action on adipocytes, accounting for reduced adiposity. Combined analysis of murine models, isolated ECs and human specimens reveals that WAT lipolysis is mediated by mTORC1-dependent production of polyamines by ECs. Our results indicate that angiocrine metabolic signals are important for WAT homeostasis and organismal metabolism.
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Adiposidad , Células Endoteliales , Animales , Células Endoteliales/metabolismo , Ratones , Ratones Endogámicos C57BL , Obesidad/metabolismo , PoliaminasRESUMEN
Opticin is an extracellular glycoprotein present in the vitreous. Its antiangiogenic properties offer the potential for therapeutic intervention in conditions such as proliferative diabetic retinopathy and retinopathy of prematurity. Here, we investigated the hypothesis that intravitreal administration of recombinant human opticin can safely protect against the development of pathological angiogenesis and promote its regression. We generated and purified recombinant human opticin and investigated its impact on the development and regression of pathological retinal neovascularization following intravitreal administration in murine oxygen-induced retinopathy. We also investigated its effect on normal retinal vascular development and function, following intravitreal injection in neonatal mice, by histological examination and electroretinography. In oxygen-induced retinopathy, intravitreal administration of human recombinant opticin protected against the development of retinal neovascularization to similar extent as aflibercept, which targets VEGF. Opticin also accelerated regression of established retinal neovascularization, though the effect at 18 h was less than that of aflibercept. Intravitreal administration of human recombinant opticin in neonatal mice caused no detectable perturbation of subsequent retinal vascular development or function. In summary we found that intraocular administration of recombinant human opticin protects against the development of pathological angiogenesis in mice and promotes its regression.
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Hiperoxia , Neovascularización Retiniana , Retinopatía de la Prematuridad , Animales , Modelos Animales de Enfermedad , Humanos , Hiperoxia/complicaciones , Recién Nacido , Inyecciones Intravítreas , Ratones , Ratones Endogámicos C57BL , Neovascularización Patológica , Oxígeno/toxicidad , Neovascularización Retiniana/tratamiento farmacológico , Retinopatía de la Prematuridad/tratamiento farmacológico , Retinopatía de la Prematuridad/prevención & controlRESUMEN
BACKGROUND: Pericytes regulate vessel stabilization and function, and their loss is associated with diseases such as diabetic retinopathy or cancer. Despite their physiological importance, pericyte function and molecular regulation during angiogenesis remain poorly understood. METHODS: To decipher the transcriptomic programs of pericytes during angiogenesis, we crossed Pdgfrb(BAC)-CreERT2 mice into RiboTagflox/flox mice. Pericyte morphological changes were assessed in mural cell-specific R26-mTmG reporter mice, in which low doses of tamoxifen allowed labeling of single-cell pericytes at high resolution. To study the role of phosphoinositide 3-kinase (PI3K) signaling in pericyte biology during angiogenesis, we used genetic mouse models that allow selective inactivation of PI3Kα and PI3Kß isoforms and their negative regulator phosphate and tensin homolog deleted on chromosome 10 (PTEN) in mural cells. RESULTS: At the onset of angiogenesis, pericytes exhibit molecular traits of cell proliferation and activated PI3K signaling, whereas during vascular remodeling, pericytes upregulate genes involved in mature pericyte cell function, together with a remarkable decrease in PI3K signaling. Immature pericytes showed stellate shape and high proliferation, and mature pericytes were quiescent and elongated. Unexpectedly, we demonstrate that PI3Kß, but not PI3Kα, regulates pericyte proliferation and maturation during vessel formation. Genetic PI3Kß inactivation in pericytes triggered early pericyte maturation. Conversely, unleashing PI3K signaling by means of PTEN deletion delayed pericyte maturation. Pericyte maturation was necessary to undergo vessel remodeling during angiogenesis. CONCLUSIONS: Our results identify new molecular and morphological traits associated with pericyte maturation and uncover PI3Kß activity as a checkpoint to ensure appropriate vessel formation. In turn, our results may open new therapeutic opportunities to regulate angiogenesis in pathological processes through the manipulation of pericyte PI3Kß activity.
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Neovascularización Fisiológica , Pericitos/enzimología , Fosfatidilinositol 3-Quinasas/metabolismo , Transducción de Señal , Remodelación Vascular , Animales , Ratones , Ratones Transgénicos , Fosfohidrolasa PTEN/genética , Fosfohidrolasa PTEN/metabolismo , Fosfatidilinositol 3-Quinasas/genéticaRESUMEN
The retinal vasculature is tightly organized in a structure that provides for the high metabolic demand of neurons while minimizing interference with incident light. The adverse impact of retinal vascular insufficiency is mitigated by adaptive vascular regeneration but exacerbated by pathological neovascularization. Aberrant growth of neovessels in the retina is responsible for impairment of sight in common blinding disorders including retinopathy of prematurity, proliferative diabetic retinopathy, and age-related macular degeneration. Myeloid cells are key players in this process, with diverse roles that can either promote or protect against ocular neovascularization. We have previously demonstrated that myeloid-derived VEGF, HIF1, and HIF2 are not essential for pathological retinal neovascularization. Here, however, we show by cell-specific depletion of Vhl in a mouse model of retinal ischemia (oxygen-induced retinopathy, OIR) that myeloid-derived HIFs promote VEGF and bFGF expression and enhance vascular regeneration in association with improved density and organization of the astrocytic network.
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Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Isquemia/genética , Células Mieloides/metabolismo , Regeneración/genética , Vasos Retinianos/fisiología , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/genética , Animales , Animales Recién Nacidos , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Hipoxia de la Célula/genética , Retinopatía Diabética/genética , Retinopatía Diabética/metabolismo , Retinopatía Diabética/patología , Modelos Animales de Enfermedad , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Isquemia/metabolismo , Isquemia/patología , Ratones , Ratones Transgénicos , Retina/patología , Enfermedades de la Retina/genética , Enfermedades de la Retina/metabolismo , Enfermedades de la Retina/patología , Factor A de Crecimiento Endotelial Vascular/genética , Factor A de Crecimiento Endotelial Vascular/metabolismo , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/metabolismoRESUMEN
Retinal ischemia and pathological angiogenesis cause severe impairment of sight. Oxygen-induced retinopathy (OIR) in young mice is widely used as a model to investigate the underlying pathological mechanisms and develop therapeutic interventions. We compared directly the conventional OIR model (exposure to 75% O2 from postnatal day (P) 7 to P12) with an alternative, accelerated version (85% O2 from P8 to P11). We found that accelerated OIR induces similar pre-retinal neovascularization but greater retinal vascular regression that recovers more rapidly. The extent of retinal gliosis is similar but neuroretinal function, as measured by electroretinography, is better maintained in the accelerated model. We found no systemic or maternal morbidity in either model. Accelerated OIR offers a safe, reliable and more rapid alternative model in which pre-retinal neovascularization is similar but retinal vascular regression is greater.
Asunto(s)
Neovascularización Retiniana/etiología , Animales , Modelos Animales de Enfermedad , Regulación hacia Abajo , Femenino , Humanos , Hiperoxia/complicaciones , Isquemia/complicaciones , Isquemia/patología , Isquemia/fisiopatología , Masculino , Ratones , Ratones Endogámicos C57BL , Regeneración , Enfermedades de la Retina/etiología , Enfermedades de la Retina/patología , Enfermedades de la Retina/fisiopatología , Neovascularización Retiniana/patología , Neovascularización Retiniana/fisiopatología , Vasos Retinianos/patología , Vasos Retinianos/fisiopatología , Factor A de Crecimiento Endotelial Vascular/genéticaRESUMEN
Gene therapy is a promising therapeutic alternative for Lysosomal Storage Disorders (LSD), as it is not necessary to correct the genetic defect in all cells of an organ to achieve therapeutically significant levels of enzyme in body fluids, from which non-transduced cells can uptake the protein correcting their enzymatic deficiency. Animal models are instrumental in the development of new treatments for LSD. Here we report the generation of the first mouse model of the LSD Muccopolysaccharidosis Type IIID (MPSIIID), also known as Sanfilippo syndrome type D. This autosomic recessive, heparan sulphate storage disease is caused by deficiency in N-acetylglucosamine 6-sulfatase (GNS). Mice deficient in GNS showed lysosomal storage pathology and loss of lysosomal homeostasis in the CNS and peripheral tissues, chronic widespread neuroinflammation, reduced locomotor and exploratory activity and shortened lifespan, a phenotype that closely resembled human MPSIIID. Moreover, treatment of the GNS-deficient animals with GNS-encoding adeno-associated viral (AAV) vectors of serotype 9 delivered to the cerebrospinal fluid completely corrected pathological storage, improved lysosomal functionality in the CNS and somatic tissues, resolved neuroinflammation, restored normal behaviour and extended lifespan of treated mice. Hence, this work represents the first step towards the development of a treatment for MPSIIID.
Asunto(s)
Terapia Genética , Enfermedades por Almacenamiento Lisosomal/terapia , Mucopolisacaridosis III/terapia , Sulfatasas/genética , Animales , Dependovirus/genética , Modelos Animales de Enfermedad , Vectores Genéticos/genética , Vectores Genéticos/uso terapéutico , Humanos , Enfermedades por Almacenamiento Lisosomal/genética , Enfermedades por Almacenamiento Lisosomal/patología , Ratones , Mucopolisacaridosis III/genética , Mucopolisacaridosis III/patología , Fenotipo , Sulfatasas/administración & dosificaciónRESUMEN
Mucopolysaccharidosis type IIIC (MPSIIIC) is a severe lysosomal storage disease caused by deficiency in activity of the transmembrane enzyme heparan-α-glucosaminide N-acetyltransferase (HGSNAT) that catalyses the N-acetylation of α-glucosamine residues of heparan sulfate. Enzyme deficiency causes abnormal substrate accumulation in lysosomes, leading to progressive and severe neurodegeneration, somatic pathology and early death. There is no cure for MPSIIIC, and development of new therapies is challenging because of the unfeasibility of cross-correction. In this study, we generated a new mouse model of MPSIIIC by targeted disruption of the Hgsnat gene. Successful targeting left LacZ expression under control of the Hgsnat promoter, allowing investigation into sites of endogenous expression, which was particularly prominent in the CNS, but was also detectable in peripheral organs. Signs of CNS storage pathology, including glycosaminoglycan accumulation, lysosomal distension, lysosomal dysfunction and neuroinflammation were detected in 2-month-old animals and progressed with age. Glycosaminoglycan accumulation and ultrastructural changes were also observed in most somatic organs, but lysosomal pathology seemed most severe in liver. Furthermore, HGSNAT-deficient mice had altered locomotor and exploratory activity and shortened lifespan. Hence, this animal model recapitulates human MPSIIIC and provides a useful tool for the study of disease physiopathology and the development of new therapeutic approaches.
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Progresión de la Enfermedad , Mucopolisacaridosis III/patología , Acetiltransferasas/deficiencia , Acetiltransferasas/metabolismo , Animales , Conducta Animal , Encéfalo/enzimología , Encéfalo/patología , Modelos Animales de Enfermedad , Glicosaminoglicanos/metabolismo , Homeostasis , Humanos , Inflamación/patología , Longevidad , Lisosomas/metabolismo , Lisosomas/patología , Lisosomas/ultraestructura , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microglía/patología , Mucopolisacaridosis III/enzimología , Especificidad de Órganos , Análisis de SupervivenciaRESUMEN
OBJECTIVE: Ocular neovascularization (ONV) is a pathological feature of sight-threatening human diseases, such as diabetic retinopathy and age-related macular degeneration. Macrophage depletion in mouse models of ONV reduces the formation of pathological blood vessels, and myeloid cells are widely considered an important source of the vascular endothelial growth factor A (VEGF). However, the importance of VEGF or its upstream regulators hypoxia-inducible factor-1α (HIF1α) and hypoxia-inducible factor-2α (HIF2α) as myeloid-derived regulators of ONV remains to be determined. APPROACH AND RESULTS: We used 2 mouse models of ONV, choroidal neovascularization and oxygen-induced retinopathy, to show that Vegfa is highly expressed by several cell types, but not myeloid cells during ONV. Moreover, myeloid-specific VEGF ablation did not reduce total ocular VEGF during choroidal neovascularization or oxygen-induced retinopathy. In agreement, the conditional inactivation of Vegfa, Hif1a, or Epas1 in recruited and resident myeloid cells that accumulated at sites of neovascularization did not significantly reduce choroidal neovascularization or oxygen-induced retinopathy. CONCLUSIONS: The finding that myeloid cells are not a significant local source of VEGF in these rodent models of ONV suggests that myeloid function in neovascular eye disease differs from skin wound healing and other neovascular pathologies.
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Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Neovascularización Coroidal/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Células Mieloides/metabolismo , Neovascularización Retiniana/metabolismo , Vasos Retinianos/metabolismo , Retinopatía de la Prematuridad/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Animales , Animales Recién Nacidos , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/deficiencia , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Neovascularización Coroidal/genética , Neovascularización Coroidal/patología , Modelos Animales de Enfermedad , Subunidad alfa del Factor 1 Inducible por Hipoxia/deficiencia , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Ratones Noqueados , Células Mieloides/patología , Oxígeno , Neovascularización Retiniana/inducido químicamente , Neovascularización Retiniana/genética , Neovascularización Retiniana/patología , Vasos Retinianos/patología , Retinopatía de la Prematuridad/inducido químicamente , Retinopatía de la Prematuridad/genética , Retinopatía de la Prematuridad/patología , Transducción de Señal , Factor A de Crecimiento Endotelial Vascular/deficiencia , Factor A de Crecimiento Endotelial Vascular/genéticaRESUMEN
Proliferative retinopathies are the leading causes of blindness in Western societies. The development of new, more efficacious treatments that take advantage of recent advances in the fields of gene and cell therapy requires further investigations on the mechanisms underlying disease onset and progression, and adequate animal models that recapitulate the pathogenesis of human proliferative retinopathy and allow evaluation of the long-term therapeutic benefits that these therapies can offer. Unfortunately, most models of retinal neovascularization have short-term evolution and diabetic rodents show a very mild retinal phenotype, limited to non-proliferative changes, and do not develop proliferative retinopathy at all. Transgenic mice overexpressing Insulin-like Growth Factor-I (IGF-I) in the retina (TgIGF-I) constitute the only rodent model currently available that develops most of the retinal alterations observed in diabetic eyes, with a temporal evolution that resembles that of the human disease. TgIGF-I have retinal vascular alterations that progress as animals age from non-proliferative to proliferative disease, making these mice an excellent model of proliferative retinopathy that, due to its slow progression, allows long-term evaluation of novel antiangiogenic therapies. At the molecular level, transgenic retinas recapitulate a variety of changes that are also observed in diabetic retinas, which reinforces the validity of this model. In addition to vascular and glial alterations, Tg-IGF-I mice show progressive neurodegeneration that leads to blindness in old animals. Thus, TgIGF-I are a useful model for testing the long-term efficacy and safety of innovative antiangiogenic, glial-modulating and neuroprotective therapies for the treatment of diabetic retinopathy and other retinal proliferative disorders.
Asunto(s)
Modelos Animales de Enfermedad , Neovascularización Retiniana , Animales , HumanosRESUMEN
Gene therapy is an attractive tool for the treatment of monogenic disorders, in particular for lysosomal storage diseases (LSD) caused by deficiencies in secretable lysosomal enzymes in which neither full restoration of normal enzymatic activity nor transduction of all affected cells are necessary. However, some LSD such as Mucopolysaccharidosis Type IIIB (MPSIIIB) are challenging because the disease's main target organ is the brain and enzymes do not efficiently cross the blood-brain barrier even if present at very high concentration in circulation. To overcome these limitations, we delivered AAV9 vectors encoding for α-N-acetylglucosaminidase (NAGLU) to the Cerebrospinal Fluid (CSF) of MPSIIIB mice with the disease already detectable at biochemical, histological and functional level. Restoration of enzymatic activity in Central Nervous System (CNS) resulted in normalization of glycosaminoglycan content and lysosomal physiology, resolved neuroinflammation and restored the pattern of gene expression in brain similar to that of healthy animals. Additionally, transduction of the liver due to passage of vectors to the circulation led to whole-body disease correction. Treated animals also showed reversal of behavioural deficits and extended lifespan. Importantly, when the levels of enzymatic activity were monitored in the CSF of dogs following administration of canine NAGLU-coding vectors to animals that were either naïve or had pre-existing immunity against AAV9, similar levels of activity were achieved, suggesting that CNS efficacy would not be compromised in patients seropositive for AAV9. Our studies provide a strong rationale for the clinical development of this novel therapeutic approach as the treatment for MPSIIIB.
Asunto(s)
Acetilglucosaminidasa/genética , Terapia Genética/métodos , Mucopolisacaridosis III/genética , Mucopolisacaridosis III/terapia , Acetilglucosaminidasa/líquido cefalorraquídeo , Animales , Encéfalo/metabolismo , Encéfalo/patología , Dependovirus/genética , Dependovirus/metabolismo , Femenino , Vectores Genéticos/genética , Vectores Genéticos/metabolismo , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Mucopolisacaridosis III/líquido cefalorraquídeo , Mucopolisacaridosis III/enzimologíaRESUMEN
For most lysosomal storage diseases (LSDs) affecting the CNS, there is currently no cure. The BBB, which limits the bioavailability of drugs administered systemically, and the short half-life of lysosomal enzymes, hamper the development of effective therapies. Mucopolysaccharidosis type IIIA (MPS IIIA) is an autosomic recessive LSD caused by a deficiency in sulfamidase, a sulfatase involved in the stepwise degradation of glycosaminoglycan (GAG) heparan sulfate. Here, we demonstrate that intracerebrospinal fluid (intra-CSF) administration of serotype 9 adenoassociated viral vectors (AAV9s) encoding sulfamidase corrects both CNS and somatic pathology in MPS IIIA mice. Following vector administration, enzymatic activity increased throughout the brain and in serum, leading to whole body correction of GAG accumulation and lysosomal pathology, normalization of behavioral deficits, and prolonged survival. To test this strategy in a larger animal, we treated beagle dogs using intracisternal or intracerebroventricular delivery. Administration of sulfamidase-encoding AAV9 resulted in transgenic expression throughout the CNS and liver and increased sulfamidase activity in CSF. High-titer serum antibodies against AAV9 only partially blocked CSF-mediated gene transfer to the brains of dogs. Consistently, anti-AAV antibody titers were lower in CSF than in serum collected from healthy and MPS IIIA-affected children. These results support the clinical translation of this approach for the treatment of MPS IIIA and other LSDs with CNS involvement.
RESUMEN
Insulin-like growth factor I (IGF-I) exerts multiple effects on different retinal cell types in both physiological and pathological conditions. Despite the growth factor's extensively described neuroprotective actions, transgenic mice with increased intraocular levels of IGF-I showed progressive impairment of electroretinographic amplitudes up to complete loss of response, with loss of photoreceptors and bipolar, ganglion, and amacrine neurons. Neurodegeneration was preceded by the overexpression of genes related to retinal stress, acute-phase response, and gliosis, suggesting that IGF-I altered normal retinal homeostasis. Indeed, gliosis and microgliosis were present from an early age in transgenic mice, before other alterations occurred, and were accompanied by signs of oxidative stress and impaired glutamate recycling. Older mice also showed overproduction of pro-inflammatory cytokines. Our results suggest that, when chronically increased, intraocular IGF-I is responsible for the induction of deleterious cellular processes that can lead to neurodegeneration, and they highlight the importance that this growth factor may have in the pathogenesis of conditions such as ischemic or diabetic retinopathy.
Asunto(s)
Gliosis/metabolismo , Factor I del Crecimiento Similar a la Insulina/metabolismo , Enfermedades Neurodegenerativas/metabolismo , Células Fotorreceptoras de Vertebrados/metabolismo , Enfermedades de la Retina/metabolismo , Células Amacrinas/metabolismo , Células Amacrinas/patología , Animales , Apoptosis , Citocinas/metabolismo , Modelos Animales de Enfermedad , Electrorretinografía , Ácido Glutámico/metabolismo , Factor I del Crecimiento Similar a la Insulina/genética , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Microglía/patología , Enfermedades Neurodegenerativas/patología , Análisis de Secuencia por Matrices de Oligonucleótidos , Estrés Oxidativo , Células Fotorreceptoras de Vertebrados/patología , Retina/metabolismo , Retina/patología , Retina/fisiopatología , Transducción de Señal , Técnicas de Cultivo de Tejidos , TranscriptomaRESUMEN
Mucopolysaccharidosis type IIIA (MPSIIIA) is a rare lysosomal storage disorder caused by mutations in the sulfamidase gene. Accumulation of glycosaminoglycan (GAG) inside the lysosomes is associated with severe neurodegeneration as well as peripheral organ pathological changes leading to death of affected individuals during adolescence. There is no cure for MPSIIIA. Due to the limitation of the blood-brain barrier, enzyme replacement therapy and gene therapy strategies attempted thus far have not achieved whole-body correction of the disease. After the systemic administration of an adeno-associated virus 9 (AAV9) vector encoding for sulfamidase under the control of a ubiquitous promoter, we were able to obtain widespread expression of the therapeutic transgene in brain and in peripheral organs, and sulfamidase activity in serum of both male and female MPSIIIA mice. This was accompanied by the normalization of GAG storage levels in most peripheral organs. In brain, decrease in GAG tissue content following AAV9 gene transfer of sulfamidase was associated with the resolution of neuroinflammation. Finally, correction of disease phenotype resulted in a remarkable prolongation of survival of both male and female AAV-treated MPSIIIA mice. This proof-of-concept study will be relevant to the future development of therapies for MPSIIIA.
Asunto(s)
Terapia Genética/métodos , Vectores Genéticos/farmacología , Glicosaminoglicanos/metabolismo , Hidrolasas/genética , Mucopolisacaridosis III/terapia , Animales , Sistema Nervioso Central/metabolismo , Dependovirus/genética , Modelos Animales de Enfermedad , Femenino , Expresión Génica , Vectores Genéticos/administración & dosificación , Hígado/metabolismo , Lisosomas/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Mucopolisacaridosis III/genética , Mucopolisacaridosis III/patología , TransgenesRESUMEN
Neovascularization associated with diabetic retinopathy (DR) and other ocular disorders is a leading cause of visual impairment and adult-onset blindness. Currently available treatments are merely palliative and offer temporary solutions. Here, we tested the efficacy of antiangiogenic gene transfer in an animal model that mimics the chronic progression of human DR. Adeno-associated viral (AAV) vectors of serotype 2 coding for antiangiogenic Pigment Epithelium Derived Factor (PEDF) were injected in the vitreous of a 1.5 month-old transgenic model of retinopathy that develops progressive neovascularization. A single intravitreal injection led to long-term production of PEDF and to a striking inhibition of intravitreal neovascularization, normalization of retinal capillary density, and prevention of retinal detachment. This was parallel to a reduction in the intraocular levels of Vascular Endothelial Growth Factor (VEGF). Normalization of VEGF was consistent with a downregulation of downstream effectors of angiogenesis, such as the activity of Matrix Metalloproteinases (MMP) 2 and 9 and the content of Connective Tissue Growth Factor (CTGF). These results demonstrate long-term efficacy of AAV-mediated PEDF overexpression in counteracting retinal neovascularization in a relevant animal model, and provides evidence towards the use of this strategy to treat angiogenesis in DR and other chronic proliferative retinal disorders.
Asunto(s)
Envejecimiento/patología , Retinopatía Diabética/patología , Retinopatía Diabética/prevención & control , Proteínas del Ojo/metabolismo , Factores de Crecimiento Nervioso/metabolismo , Retina/metabolismo , Neovascularización Retiniana/patología , Neovascularización Retiniana/prevención & control , Serpinas/metabolismo , Animales , Hipoxia de la Célula , Dependovirus/genética , Retinopatía Diabética/metabolismo , Modelos Animales de Enfermedad , Regulación hacia Abajo , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Factor I del Crecimiento Similar a la Insulina/metabolismo , Inyecciones Intravítreas , Ratones , Ratones Transgénicos , Retina/patología , Desprendimiento de Retina/metabolismo , Desprendimiento de Retina/patología , Neovascularización Retiniana/metabolismo , Factores de Tiempo , Transducción Genética , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Mucopolysaccharidosis type IIIA (MPSIIIA) is an inherited lysosomal storage disease caused by deficiency of sulfamidase, resulting in accumulation of the glycosaminoglycan (GAG) heparan sulfate. It is characterized by severe progressive neurodegeneration, together with somatic alterations, which lead to death during adolescence. Here, we tested the ability of adeno-associated virus (AAV) vector-mediated genetic modification of either skeletal muscle or liver to revert the already established disease phenotype of 2-month-old MPSIIIA males and females. Intramuscular administration of AAV-Sulfamidase failed to achieve significant therapeutic benefit in either gender. In contrast, AAV8-mediated liver-directed gene transfer achieved high and sustained levels of circulating active sulfamidase, which reached normal levels in females and was fourfold higher in males, and completely corrected lysosomal GAG accumulation in most somatic tissues. Remarkably, a 50% reduction of GAG accumulation was achieved throughout the entire brain of males, which correlated with a partial improvement of the pathology of cerebellum and cortex. Liver-directed gene transfer expanded the lifespan of MPSIIIA males, underscoring the importance of reaching supraphysiological plasma levels of enzyme for maximal therapeutic benefit. These results show how liver-directed gene transfer can reverse somatic and ameliorate neurological pathology in MPSIIIA.
Asunto(s)
Sistema Nervioso Central/patología , Terapia Genética , Hidrolasas/genética , Hígado/metabolismo , Mucopolisacaridosis III/terapia , Animales , Cerebelo/ultraestructura , Dependovirus/genética , Modelos Animales de Enfermedad , Femenino , Orden Génico , Técnicas de Transferencia de Gen , Vectores Genéticos/administración & dosificación , Vectores Genéticos/genética , Vectores Genéticos/farmacocinética , Hidrolasas/metabolismo , Inyecciones Intramusculares , Inyecciones Intravenosas , Hígado/ultraestructura , Lisosomas/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mucopolisacaridosis III/genética , Mucopolisacaridosis III/mortalidad , Músculo Esquelético/metabolismo , Análisis de Supervivencia , Transducción Genética , Corteza Visual/patología , Corteza Visual/ultraestructuraRESUMEN
Blood-retinal barrier (BRB) breakdown is a key event in diabetic retinopathy and other ocular disorders that leads to increased retinal vascular permeability. This causes edema and tissue damage resulting in visual impairment. Insulin-like growth factor-I (IGF-I) is involved in these processes, although the relative contribution of increased systemic versus intraocular IGF-I remains controversial. Here, to elucidate the role of this factor in BRB breakdown, transgenic mice with either local or systemic elevations of IGF-I have been examined. High intraocular IGF-I, resulting from overexpression of IGF-I in the retina, increased IGF-I receptor content and signaling and led to accumulation of vascular endothelial growth factor. This was parallel to up-regulation of vascular Intercellular adhesion molecule I and retinal infiltration by bone marrow-derived microglial cells. These alterations resulted in increased vessel paracellular permeability to both low and high molecular weight compounds in IGF-I-overexpressing retinas and agreed with the loss of vascular tight junction integrity observed by electron microscopy and the altered junctional protein content. In contrast, mice with chronically elevated serum IGF-I did not show alterations in the retinal vasculature structure and permeability, indicating that circulating IGF-I cannot initiate BRB breakdown. Consistent with a key role of IGF-I signaling in retinal diseases, a strong up-regulation of the IGF-I receptor in human retinas with marked gliosis was also observed. Thus, this study demonstrates that intraocular IGF-I, but not systemic IGF-I, is sufficient to trigger processes leading to BRB breakdown and increased retinal vascular permeability. Therefore, therapeutic interventions designed to counteract local IGF-I effects may prove successful to prevent BRB disruption.