RESUMEN

Autosomal dominant optic atrophy (ADOA) is an inherited optic neuropathy most frequently associated with OPA1 mutations. Most variants result in haploinsufficiency, and patient cells express roughly half of the normal levels of OPA1 protein. OPA1 is a mitochondrial GTPase that is essential for normal mitochondrial function. We identified and characterized STK-002, an antisense oligonucleotide (ASO) designed to prevent the incorporation of a naturally occurring alternatively spliced nonproductive exon in OPA1. STK-002 dose dependently reduced the inclusion of this exon, and increased OPA1 protein in human cells, including ADOA patient-derived fibroblasts. ADOA patient cells manifest reduced mitochondrial respiration, and treatment with STK-002 improved the parameters of mitochondrial respiratory function in these cells. Since STK-002 increases OPA1 through the wild-type allele, we assessed retinal OPA1 in wild-type cynomolgus monkeys and rabbits after intravitreal administration of STK-002 or a rabbit-specific surrogate. Increased OPA1 protein was produced in retinal tissue in both species at 4 weeks after ASO injection and persisted in monkeys at 8 weeks. STK-002 and enhanced OPA1 immunofluorescence were visualized in retinal ganglion cells of cynomolgus monkeys treated with the ASO. Cumulatively, these data support the progression of STK-002 toward the clinic as the first potential disease-modifying treatment for ADOA.

RESUMEN

Dravet Syndrome (DS) is a severe developmental and epileptic encephalopathy typically caused by loss-of-function de novo mutations in the SCN1A gene which encodes the voltage-gated sodium channel isoform NaV1.1. Decreased NaV1.1 expression results in impaired excitability of inhibitory interneurons and seizure onset. To date, there are no clinically available treatments for DS that directly address the core mechanism of disease; reduced NaV1.1 expression levels in interneurons. Recently, Targeted Augmentation of Nuclear Gene Output (TANGO) of SCN1A by the antisense oligonucleotide (ASO) STK-001, was shown to increase Scn1a mRNA levels, increase NaV1.1 protein expression, reduce seizures, and improve survival in the Scn1a+/- mouse model of DS. However, it remains unknown whether STK-001 treatment rescues the reduced intrinsic excitability of parvalbumin-positive (PV) inhibitory interneurons associated with DS. In this study, we demonstrate that STK-001 treatment reduces seizures, prolongs survival, and rescues PV interneuron excitability in Scn1a+/- mice to levels observed in WT littermates. Together, these results support the notion that TANGO-mediated augmentation of NaV1.1 levels directly targets and rescues one of the core disease mechanisms of DS.

Asunto(s)

Potenciales de Acción/fisiología , Epilepsias Mioclónicas/genética , Interneuronas/metabolismo , Canal de Sodio Activado por Voltaje NAV1.1/genética , Parvalbúminas/metabolismo , Convulsiones/genética , Animales , Modelos Animales de Enfermedad , Epilepsias Mioclónicas/fisiopatología , Ratones , Oligonucleótidos Antisentido , Convulsiones/fisiopatología

RESUMEN

Densitometric analysis is often used to quantify NaV1.1 protein on immunoblots, although the sensitivity and dilution linearity of the method are usually poor. Here we present a protocol for quantification of NaV1.1 in mouse brain tissues using a Meso Scale Discovery-Electrochemiluminescence (MSD-ECL) method. MSD-ECL is based on ELISA (enzyme-linked immunosorbent assay) and uses electrochemiluminescence to produce measurable signals. Two different antibodies are used in this assay to capture and detect NaV1.1 respectively in brain tissue lysate. The specificity of the antibodies is confirmed by Scn1a gene knock-out tissue. The calibration curve standards used in this assay were generated with mouse liver lysate spiked with mouse brain lysate, instead of using a recombinant protein. We showed that this method was qualified and used for quantification of NaV1.1 in mouse brain tissues with specificity, accuracy and precision.

RESUMEN

Dravet syndrome (DS) is an intractable developmental and epileptic encephalopathy caused largely by de novo variants in the SCN1A gene, resulting in haploinsufficiency of the voltage-gated sodium channel α subunit NaV1.1. Here, we used Targeted Augmentation of Nuclear Gene Output (TANGO) technology, which modulates naturally occurring, nonproductive splicing events to increase target gene and protein expression and ameliorate disease phenotype in a mouse model. We identified antisense oligonucleotides (ASOs) that specifically increase the expression of productive Scn1a transcript in human cell lines, as well as in mouse brain. We show that a single intracerebroventricular dose of a lead ASO at postnatal day 2 or 14 reduced the incidence of electrographic seizures and sudden unexpected death in epilepsy (SUDEP) in the F1:129S-Scn1a +/- × C57BL/6J mouse model of DS. Increased expression of productive Scn1a transcript and NaV1.1 protein was confirmed in brains of treated mice. Our results suggest that TANGO may provide a unique, gene-specific approach for the treatment of DS.

Asunto(s)

Epilepsias Mioclónicas , Muerte Súbita e Inesperada en la Epilepsia , Animales , Epilepsias Mioclónicas/genética , Incidencia , Ratones , Ratones Endogámicos C57BL , Canal de Sodio Activado por Voltaje NAV1.1/genética , Oligonucleótidos Antisentido , Convulsiones/genética

RESUMEN

While most monogenic diseases are caused by loss or reduction of protein function, the need for technologies that can selectively increase levels of protein in native tissues remains. Here we demonstrate that antisense-mediated modulation of pre-mRNA splicing can increase endogenous expression of full-length protein by preventing naturally occurring non-productive alternative splicing and promoting generation of productive mRNA. Bioinformatics analysis of RNA sequencing data identifies non-productive splicing events in 7,757 protein-coding human genes, of which 1,246 are disease-associated. Antisense oligonucleotides targeting multiple types of non-productive splicing events lead to increases in productive mRNA and protein in a dose-dependent manner in vitro. Moreover, intracerebroventricular injection of two antisense oligonucleotides in wild-type mice leads to a dose-dependent increase in productive mRNA and protein in the brain. The targeting of natural non-productive alternative splicing to upregulate expression from wild-type or hypomorphic alleles provides a unique approach to treating genetic diseases.

Asunto(s)

Empalme Alternativo , Regulación de la Expresión Génica , Oligonucleótidos Antisentido/farmacología , Alelos , Animales , Animales Recién Nacidos , Encéfalo/metabolismo , Biología Computacional , Exones , Femenino , Expresión Génica/efectos de los fármacos , Células HEK293 , Humanos , Intrones , Masculino , Ratones , Ratones Endogámicos C57BL , ARN Mensajero/metabolismo , Activación Transcripcional/efectos de los fármacos , Regulación hacia Arriba

RESUMEN

BACKGROUND AND PURPOSE: Inhibition of the renin angiotensin system (RAS) has been consistently demonstrated to reduce atherosclerosis. However, there has been no direct comparison among the three available pharmacological modes of inhibiting the RAS, which are inhibitors of renin, ACE and angiotensin II type 1 receptor. The purpose of this study was to determine the relative effects of these three modes of pharmacological RAS inhibition in reducing atherosclerosis by determining the dose-response relationships. EXPERIMENTAL APPROACH: Male LDL receptor -/- mice were administered either vehicle or any of three doses of aliskiren, enalapril or losartan through s.c. infusion for 12 weeks. All mice were fed a saturated fat-enriched diet during drug infusions. Systolic and diastolic BPs were measured during the study using a non-invasive tail-cuff system. Plasma cholesterol and renin concentrations, atherosclerotic lesions, and renal angiotensin II concentrations were determined at the termination of the study. KEY RESULTS: Plasma renin concentrations were increased by all three drugs. None of the drugs changed plasma cholesterol concentrations. All drugs produced a dose-related decrease in BP. All three drugs also profoundly reduced atherosclerosis in a dose-dependent manner. The highest dose of each drug markedly attenuated lesion size, with no significant differences between the different drugs. The highest dose of each drug also similarly reduced renal angiotensin II concentrations. CONCLUSION AND IMPLICATIONS: Drugs that inhibit the RAS, irrespective of their mode of inhibition, profoundly affect atherosclerotic lesion development in a dose-dependent manner.

Asunto(s)

Bloqueadores del Receptor Tipo 1 de Angiotensina II/uso terapéutico , Inhibidores de la Enzima Convertidora de Angiotensina/uso terapéutico , Aterosclerosis/tratamiento farmacológico , Hipercolesterolemia/tratamiento farmacológico , Renina/antagonistas & inhibidores , Amidas/farmacología , Amidas/uso terapéutico , Angiotensina II/metabolismo , Bloqueadores del Receptor Tipo 1 de Angiotensina II/farmacología , Inhibidores de la Enzima Convertidora de Angiotensina/farmacología , Animales , Aorta/patología , Aterosclerosis/etiología , Aterosclerosis/patología , Aterosclerosis/fisiopatología , Presión Sanguínea/efectos de los fármacos , Colesterol/sangre , Dieta Alta en Grasa , Enalapril/farmacología , Enalapril/uso terapéutico , Fumaratos/farmacología , Fumaratos/uso terapéutico , Hipercolesterolemia/complicaciones , Hipercolesterolemia/etiología , Hipercolesterolemia/fisiopatología , Riñón/metabolismo , Losartán/farmacología , Masculino , Ratones , Ratones Noqueados , Receptor de Angiotensina Tipo 1/fisiología , Receptores de LDL/deficiencia , Receptores de LDL/genética , Renina/sangre , Sistema Renina-Angiotensina/efectos de los fármacos

RESUMEN

OBJECTIVE: Aliskiren is the first commercially available, orally active, direct renin inhibitor approved to treat hypertension. The renin-angiotensin system has been shown to be a significant contributor to the development of hypercholesterolemia-induced atherosclerosis. The aim of this study was to evaluate the antiatherosclerotic and plaque stabilization effects of aliskiren alone and in combination with atorvastatin. METHODS: APOE*3Leiden.CETP mice (n = 14-17/group) were fed a western-type diet (containing 0.25% cholesterol) alone or were treated with either aliskiren (15 mg/kg per day), atorvastatin (3.6 mg/kg per day) or a combination of aliskiren and atorvastatin. Effects on SBP, total cholesterol, inflammation markers and atherosclerotic size and composition were assessed. RESULTS: Aliskiren reduced SBP (-19%, P < 0.001) and atorvastatin reduced total cholesterol (-24%, P < 0.001). Atherosclerotic lesion area was reduced by aliskiren (-40%, P < 0.01), atorvastatin (-61%, P < 0.001) and the combination treatment (-69%, P < 0.001). Aliskiren alone and together with atorvastatin decreased the number of T cells in the aortic root area (-60%, P < 0.01; -41%, P < 0.05), as well as macrophage (-64%, P < 0.001; -72%, P < 0.001) and necrotic area (-52%, P = 0.071; -84%, P < 0.001) in the lesion. Atorvastatin alone and together with aliskiren decreased monocyte adherence (-43%, P < 0.05 and -51%, P < 0.01) and monocyte chemoattractant protein-1 (both -36%, P < 0.01). The combination treatment decreased the number of lesions (-17%, P < 0.05) and E-selectin (-17%, P < 0.05). CONCLUSION: Aliskiren inhibited atherosclerosis development and improved plaque stability alone and in combination with atorvastatin, possibly via a mechanism involving T cells. These results suggest a potential benefit of using aliskiren in a clinical setting, particularly in combination with statin treatment.

Asunto(s)

Amidas/farmacología , Apolipoproteínas E/genética , Aterosclerosis/prevención & control , Fumaratos/farmacología , Ácidos Heptanoicos/farmacología , Inhibidores de Hidroximetilglutaril-CoA Reductasas/farmacología , Pirroles/farmacología , Animales , Aterosclerosis/genética , Atorvastatina , Femenino , Ratones , Ratones Transgénicos

RESUMEN

PCSK9 (proprotein convertase subtilisin/kexin 9) is a secreted serine protease that regulates cholesterol homoeostasis by inducing post-translational degradation of hepatic LDL-R [LDL (low-density lipoprotein) receptor]. Intramolecular autocatalytic processing of the PCSK9 zymogen in the endoplasmic reticulum results in a tightly associated complex between the prodomain and the catalytic domain. Although the autocatalytic processing event is required for proper secretion of PCSK9, the requirement of proteolytic activity in the regulation of LDL-R is currently unknown. Co-expression of the prodomain and the catalytic domain in trans allowed for production of a catalytically inactive secreted form of PCSK9. This catalytically inactive PCSK9 was characterized and shown to be functionally equivalent to the wild-type protein in lowering cellular LDL uptake and LDL-R levels. These findings suggest that, apart from autocatalytic processing, the protease activity of PCSK9 is not necessary for LDL-R regulation.

Asunto(s)

Receptores de LDL/metabolismo , Serina Endopeptidasas/metabolismo , Línea Celular , Humanos , Mutación/genética , Serina/genética , Serina/metabolismo , Serina Endopeptidasas/genética

RESUMEN

FTY720, a potent immunomodulator, becomes phosphorylated in vivo (FTY-P) and interacts with sphingosine-1-phosphate (S1P) receptors. Recent studies showed that FTY-P affects vascular endothelial growth factor (VEGF)-induced vascular permeability, an important aspect of angiogenesis. We show here that FTY720 has antiangiogenic activity, potently abrogating VEGF- and S1P-induced angiogenesis in vivo in growth factor implant and corneal models. FTY720 administration tended to inhibit primary and significantly inhibited metastatic tumor growth in a mouse model of melanoma growth. In combination with a VEGFR tyrosine kinase inhibitor PTK787/ZK222584, FTY720 showed some additional benefit. FTY720 markedly inhibited tumor-associated angiogenesis, and this was accompanied by decreased tumor cell proliferation and increased apoptosis. In transfected HEK293 cells, FTY-P internalized S1P1 receptors, inhibited their recycling to the cell surface, and desensitized S1P receptor function. Both FTY720 and FTY-P apparently failed to impede VEGF-produced increases in mitogen-activated protein kinase activity in human umbilical vascular endothelial cells (HUVEC), and unlike its activity in causing S1PR internalization, FTY-P did not result in a decrease of surface VEGFR2 levels in HUVEC cells. Pretreatment with FTY720 or FTY-P prevented S1P-induced Ca2+ mobilization and migration in vascular endothelial cells. These data show that functional antagonism of vascular S1P receptors by FTY720 potently inhibits angiogenesis; therefore, this may provide a novel therapeutic approach for pathologic conditions with dysregulated angiogenesis.

Asunto(s)

Melanoma Experimental/irrigación sanguínea , Melanoma Experimental/tratamiento farmacológico , Glicoles de Propileno/farmacología , Receptores de Lisoesfingolípidos/antagonistas & inhibidores , Animales , Calcio/metabolismo , Procesos de Crecimiento Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Córnea/irrigación sanguínea , Células Endoteliales/efectos de los fármacos , Células Endoteliales/enzimología , Femenino , Clorhidrato de Fingolimod , Humanos , Melanoma Experimental/metabolismo , Melanoma Experimental/patología , Ratones , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Neovascularización Patológica/tratamiento farmacológico , Neovascularización Fisiológica/efectos de los fármacos , Fosforilación , Ftalazinas/farmacología , Glicoles de Propileno/farmacocinética , Piridinas/farmacología , Receptores de Lisoesfingolípidos/metabolismo , Esfingosina/análogos & derivados , Factor A de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismo

RESUMEN

OBJECTIVES: We previously demonstrated that sphingosine kinase (SPK) increases the level of extracellular sphingosine-1-phosphate and promotes neovascularization in a mouse matrigel model. In this study, we tested the hypothesis that SPK gene transfer using a novel adenoviral 'gutless' vector (AGV) can enhance arteriogenesis in a rabbit hindlimb ischemia model. METHODS: Thirty-five male New Zealand white rabbits were randomized to the AGV-SPK group (n=13), AGV-null group (n=13), and control group (n=9). On day 10, after the induction of unilateral hindlimb ischemia, gene vectors or buffer were introduced and the effect examined on day 30, using calf blood pressure, quantitative angiographic analysis, and histology. RESULTS: Calf systolic blood pressure ratios of the ischemic limb to the normal limb on day 30 were 0.77+/-0.13 in control groups, including the AGV-null group, and 0.91+/-0.14 in the AGV-SPK group (P<0.05). Angiographic vessel counts were significantly increased (8.0+/-2.1 at baseline and 11.8+/-3.2 on day 30, P<0.001) in the AGV-SPK group. Histologic analysis showed that microscopic total vessel counts on day 30 were 3.5+/-1.8/field in the control and AGV-null group and 5.4+/-1.0/field in the AGV-SPK group. Arterioles (AGV-SPK; 3.0+/-0.8 versus control and AGV-null; 2.1+/-1.1, P<0.05) were significantly increased in the AGV-SPK group. CONCLUSIONS: This study shows that SPK promotes arteriogenesis, as evidenced by the maximal improvement in the blood pressure restoration and collateral vessel counts. SPK may be an important angiogenic target to improve perfusion in ischemic tissues.

Asunto(s)

Inductores de la Angiogénesis/metabolismo , Terapia Genética/métodos , Isquemia/tratamiento farmacológico , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Adenoviridae/genética , Inductores de la Angiogénesis/uso terapéutico , Angiografía , Animales , Arteria Femoral/efectos de los fármacos , Arteria Femoral/crecimiento & desarrollo , Técnicas de Transferencia de Gen , Vectores Genéticos/biosíntesis , Miembro Posterior/irrigación sanguínea , Masculino , Modelos Animales , Neovascularización Fisiológica/efectos de los fármacos , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Fosfotransferasas (Aceptor de Grupo Alcohol)/uso terapéutico , Conejos , Flujo Sanguíneo Regional/efectos de los fármacos

RESUMEN

Vascular endothelial growth factor (VEGF) and insulin-like growth factor-1 (IGF-1) have been implicated as important stimulatory factors for retinal neovascularization. In this study, we used intraocular gene transfer with gutless adenoviral (AGV) vectors to determine the effect of increased intraocular expression of VEGF, IGF-1, or sphingosine kinase (SPK), which produces sphingosine-1-phosphate, another angiogenic factor. Retinal neovascularization did not occur from intravitreous AGV-vectored VEGF, IGF-1, SPK, or combined VEGF and IGF-1, except occasionally adjacent to the retinal penetration site from the injection. However, corneal and iris neovascularization occurred after 2 weeks in all eyes injected with AGV.VEGF, but not those injected with only AGV.IGF-1 or AGV.SPK. These data suggest that the superficial capillary bed of the retina is relatively insensitive to VEGF, IGF-1, or SPK in adult mice, except when combined with retinal trauma. However, AGV-vectored VEGF is sufficient to consistently cause severe corneal and iris neovascularization. This provides a model for anterior segment neovascularization, which unlike previous models is relatively inexpensive and is not plagued by spontaneous regression, and therefore, may be useful for identification of new treatments.

Asunto(s)

Adenoviridae , Ojo/irrigación sanguínea , Neovascularización Retiniana , Transducción Genética , Factor A de Crecimiento Endotelial Vascular/biosíntesis , Adenoviridae/genética , Animales , Cartilla de ADN , Vectores Genéticos , Humanos , Procesamiento de Imagen Asistido por Computador , Inmunohistoquímica , Factor I del Crecimiento Similar a la Insulina/biosíntesis , Factor I del Crecimiento Similar a la Insulina/genética , Operón Lac , Lisofosfolípidos/metabolismo , Ratones , Neovascularización Fisiológica/genética , Fosfotransferasas (Aceptor de Grupo Alcohol)/biosíntesis , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Esfingosina/análogos & derivados , Esfingosina/metabolismo , Factor A de Crecimiento Endotelial Vascular/genética

RESUMEN

The amino-terminal domain of the extracellular matrix (ECM) protein thrombospondin-1 (TSP-1) mediates binding to cell surface heparan sulfate proteoglycans (HSPG) as well as binding to the endocytic receptor, low density lipoprotein-related protein (LRP-1). We previously found that recombinant TSP-1 containing the amino-terminal residues 1-214, retained both of these interactions (Mikhailenko et al. [1997]: J Biol Chem 272:6784-6791). Here, we examined the activity of a recombinant protein containing amino-terminal residues 1-90 of TSP-1 and found that this domain did not retain high-affinity heparin-binding. The loss of heparin-binding correlated with decreased binding to the fibroblast cell surface. However, both ligand blotting and solid phase binding studies indicate that this truncated fragment of TSP-1 retained high-affinity binding to LRP-1. Consistent with this, it also retained the ability to block the uptake and degradation of (125)I-TSP-1. However, TSP-1(1-90) itself was poorly endocytosed and this truncated amino-terminal domain was considerably more effective than the full-length heparin-binding domain (HBD) of TSP-1 in blocking the catabolism of endogenously expressed TSP-1. These results indicate that TSP-1 binding to LRP-1 does not require prior or concomitant interaction with cell surface HSPG but suggest subsequent endocytosis requires high-affinity heparin-binding.

Asunto(s)

Endocitosis , Proteoglicanos de Heparán Sulfato/metabolismo , Proteína 1 Relacionada con Receptor de Lipoproteína de Baja Densidad/metabolismo , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Trombospondina 1/química , Trombospondina 1/metabolismo , Animales , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Medios de Cultivo Condicionados/química , Medios de Cultivo Condicionados/metabolismo , Endocitosis/efectos de los fármacos , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Proteoglicanos de Heparán Sulfato/química , Proteoglicanos de Heparán Sulfato/genética , Heparina/metabolismo , Humanos , Ratones , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/farmacología , Unión Proteica/efectos de los fármacos , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes de Fusión/farmacología , Proteínas Recombinantes/genética , Trombospondina 1/farmacología

RESUMEN

BACKGROUND: Imprinted genes, mesodermal specific cDNA or transcript (MEST) and H19, are implicated in peri-implantation embryogenesis, and their expression was assessed in embryonic kidneys undergoing glucose-induced dysmorphogenesis. METHODS: MEST and H19 mRNA expression was assessed by Northern blot analysis in embryonic kidneys of mice harvested at day 15 to day 19 of gestation and of 1-week-old mice obtained from hyperglycemic mothers. A full-length mouse MEST cDNA was isolated, subcloned into an expression vector, a recombinant protein prepared and an antibody raised; the latter was used to assess protein expression by immunoprecipitation and immunofluorescence microscopy in day 13 metanephric explants subjected to high glucose ambience. Also, MEST mRNA expression was assessed in high d glucose-treated explants by competitive reverse transcription-polymerase chain reaction (RT-PCR) analyses and by in situ tissue autoradiography. RESULTS: A high expression of MEST and H19 with respective transcript size of approximately 2.7 and approximately 2.4 kb was observed in fetal kidneys, and their expression decreased during the successive stages of gestation and was undetectable in the postnatal period. At day 13, the MEST mRNA was expressed in the mesenchyme, while H19 was expressed in the ureteric bud branches and epithelial elements of the metanephros. Their expression decreased with progression of gestation. By competitive RT-PCR and Northern blot and in situ autoradiographic analyses, both MEST and H19 expressions decreased in day 13 explants treated with high glucose and in the kidneys of fetuses obtained from diabetic mothers. The MEST protein expression was observed in the metanephric epithelial elements and ureteric bud branches instead of in the mesenchyme, and its expression decreased in glucose-treated dysmorphogenetic explants, as assessed by immunofluorescence and immunoprecipitation methods. CONCLUSION: MEST and H19 imprinted genes are strategically located in the mammalian embryonic metanephros. They are developmentally regulated and their concomitant decreased expression in high glucose ambience or diabetic state did not follow the prevailing dogma of reciprocal inactivation/activation of imprinted genes, and such a decrease may be responsible for the perturbed epithelial:mesenchymal interactions leading to dysmorphogenesis of the mammalian metanephros.

Asunto(s)

Nefropatías Diabéticas/fisiopatología , Impresión Genómica , Riñón/fisiología , Proteínas/genética , ARN no Traducido/genética , Animales , Animales Recién Nacidos , Anticuerpos/farmacología , Femenino , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Glucosa/farmacología , Hiperglucemia/fisiopatología , Riñón/embriología , Masculino , Ratones , Ratones Endogámicos ICR , Técnicas de Cultivo de Órganos , Embarazo , Embarazo en Diabéticas/fisiopatología , Proteínas/inmunología , Proteínas/farmacología , ARN Largo no Codificante , ARN Mensajero/análisis , Proteínas Recombinantes de Fusión/farmacología

RESUMEN

Endostatin, a proteolytic fragment of collagen XVIII, is an endogenous inhibitor of tumor angiogenesis that also inhibits choroidal neovascularization. In this study, we assessed the effects of increased intraocular expression of endostatin on vascular endothelial growth factor (VEGF)-induced changes in the retina. After subretinal injection of a pair of gutless adenoviral vectors (AGV) designed to provide tamoxifen-inducible expression of endostatin, diffuse endostatin immunoreactivity was induced thoroughout the retina by administration of tamoxifen. Induction of endostatin in double transgenic mice with doxycycline-induced expression of VEGF in the retina resulted in significant suppression of leakage of intravascular [3H]mannitol into the retina. The ability of endostatin to reduce VEGF-induced retinal vascular permeability was confirmed by using [3H]mannitol leakage and two other parameters, fluorescein leakage and retinal thickness, after subretinal injection of a bovine immunodeficiency lentiviral vector coding for endostatin (BIV-vectored endostatin, or BIVendostatin). Subretinal injection of BIVendostatin resulted in more discrete, less intense staining for endostatin in the retina than that seen with the inducible AGV system, which suggested lower levels and allowed visualization of sites where endostatin was concentrated. Endostatin staining outlined retinal blood vessels, which suggested endostatin binding to a component of vessel walls. More prolonged or higher level expression of VEGF in the retina resulted in neovascularization and retinal detachment, both of which were also significantly reduced by BIVendostatin. These data suggest that endostatin may be an endogenous inhibitor of vasopermeability as well as neovascularization. In patients with diabetic retinopathy, endostatin gene transfer may provide a way to decrease the risk of three causes of visual loss: macular edema, neovascularization, and retinal detachment.

Asunto(s)

Permeabilidad Capilar/efectos de los fármacos , Colágeno/fisiología , Factores de Crecimiento Endotelial/farmacología , Ojo/metabolismo , Péptidos y Proteínas de Señalización Intercelular/farmacología , Linfocinas/farmacología , Fragmentos de Péptidos/fisiología , Desprendimiento de Retina/patología , Neovascularización Retiniana/patología , Animales , Colágeno/biosíntesis , Colágeno/genética , Colágeno Tipo XVIII , Endostatinas , Factores de Crecimiento Endotelial/genética , Ojo/irrigación sanguínea , Ojo/efectos de los fármacos , Péptidos y Proteínas de Señalización Intercelular/genética , Linfocinas/genética , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Fragmentos de Péptidos/biosíntesis , Fragmentos de Péptidos/genética , Retina/efectos de los fármacos , Retina/metabolismo , Retina/patología , Desprendimiento de Retina/inducido químicamente , Neovascularización Retiniana/inducido químicamente , Transfección/métodos , Factor A de Crecimiento Endotelial Vascular , Factores de Crecimiento Endotelial Vascular

RESUMEN

The enzyme sphingosine kinase (SK) catalyzes the formation of sphingosine 1-phosphate (S1P), a bioactive lipid that acts extracellularly on G protein-coupled receptors of the S1P(1)/EDG-1 subfamily. Although S1P is formed in the cytosol of various cells, S1P release is not understood and is controversial because this lipid mediator is also regarded as a second messenger. In this report, we describe the existence of an extracellular S1P-generating system in vascular endothelial cells. Endothelial cells release SK constitutively and form S1P in the range of receptor stimulation. Levels of sphingosine but not ATP in the extracellular environment are rate-limiting. Treatment of endothelial cells with small interfering RNA for SK-1 transcript specifically inhibited SK export, and SK-1-transfected human embryonic kidney 293 cells exhibited enhanced release of SK-1. The export of SK-1 is constitutive and is inhibited by cytochalasin D and treatment at 4 degrees C but not by brefeldin A or nocodazole, suggesting that a nonclassical secretory pathway that requires the actin cytoskeleton dynamics is involved. Because S1P regulates angiogenesis and vascular maturation, we overexpressed SK-1 using an adenoviral vector in vivo in the Matrigel system of angiogenesis. Overexpression of SK-1 resulted in enhanced release of SK activity and induced angiogenesis and vascular maturation. These findings suggest that S1P is made in the extracellular milieu and that extracellular export of SK contributes to the action of S1P in the vascular system.

Asunto(s)

Endotelio Vascular/metabolismo , Regulación Enzimológica de la Expresión Génica , Lisofosfolípidos , Neovascularización Fisiológica , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Receptores de Superficie Celular , Receptores Acoplados a Proteínas G , Esfingosina/análogos & derivados , Esfingosina/metabolismo , Proteínas de Xenopus , Animales , Calcio/metabolismo , Línea Celular , Medios de Cultivo Condicionados , Citosol/metabolismo , Femenino , Silenciador del Gen , Proteínas de Homeodominio/metabolismo , Humanos , Proteínas Inmediatas-Precoces/metabolismo , Riñón , Cinética , Oocitos/fisiología , Transporte de Proteínas , ARN Interferente Pequeño , ARN no Traducido/genética , Receptores Lisofosfolípidos , Proteínas Represoras/metabolismo , Transfección , Xenopus , Caja Homeótica 2 de Unión a E-Box con Dedos de Zinc